Research

13 pages, 1586 KiB

Open AccessArticle

Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered E. coli

by Mitzi de la Cruz, Flavio Kunert, Hilal Taymaz-Nikerel, Juan-Carlos Sigala, Guillermo Gosset, Jochen Büchs and Alvaro R. Lara

Microorganisms 2024, 12(1), 150; https://doi.org/10.3390/microorganisms12010150 - 12 Jan 2024

Viewed by 1108

Abstract

The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the [...] Read more.

The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the pyruvate kinase A gene has been shown to produce more pDNA than its parental strain. With the aim of improving pDNA production in the engineered strain, several strategies to increase the flux to the pentose phosphate pathway (PPP) were evaluated. The simultaneous consumption of glucose and glycerol was a simple way to increase the growth rate, pDNA production rate, and supercoiled fraction (SCF). The overexpression of key genes from the PPP also improved pDNA production in glucose, but not in mixtures of glucose and glycerol. Particularly, the gene coding for the glucose 6-phosphate dehydrogenase (G6PDH) strongly improved the SCF, growth rate, and pDNA production rate. A linear relationship between the G6PDH activity and pDNA yield was found. A higher flux through the PPP was confirmed by flux balance analysis, which also estimates relevant differences in fluxes of the tricarboxylic acid cycle. These results are useful for developing further cell engineering strategies to increase pDNA production and quality. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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0 pages, 2813 KiB

Open AccessArticle

Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains

by Anastasiya Makeeva, Dmitry Muzaev, Maria Shubert, Tatiana Ianshina, Anton Sidorin, Elena Sambuk, Andrey Rumyantsev and Marina Padkina

Microorganisms 2023, 11(9), 2297; https://doi.org/10.3390/microorganisms11092297 - 12 Sep 2023

Viewed by 879

Abstract

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent [...] Read more.

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate Mut^S strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii Mut^S strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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23 pages, 3268 KiB

Open AccessArticle

Exploring the Influence of pH on the Dynamics of Acetone–Butanol–Ethanol Fermentation

by Manish Kumar, Supreet Saini and Kalyan Gayen

Microorganisms 2023, 11(6), 1610; https://doi.org/10.3390/microorganisms11061610 - 18 Jun 2023

Cited by 1 | Viewed by 1236

Abstract

Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its ability to produce butanol. Over the past two decades, various genetic and metabolic engineering approaches have been used to investigate the physiology and regulation system of the biphasic metabolic pathway in [...] Read more.

Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its ability to produce butanol. Over the past two decades, various genetic and metabolic engineering approaches have been used to investigate the physiology and regulation system of the biphasic metabolic pathway in this organism. However, there has been a relatively limited amount of research focused on the fermentation dynamics of C. acetobutylicum. In this study, we developed a pH-based phenomenological model to predict the fermentative production of butanol from glucose using C. acetobutylicum in a batch system. The model describes the relationship between the dynamics of growth and the production of desired metabolites and the extracellular pH of the media. Our model was found to be successful in predicting the fermentation dynamics of C. acetobutylicum, and the simulations were validated using experimental fermentation data. Furthermore, the proposed model has the potential to be extended to represent the dynamics of butanol production in other fermentation systems, such as fed-batch or continuous fermentation using single and multi-sugars. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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14 pages, 6682 KiB

Open AccessArticle

Comparative Analysis Using Raman Spectroscopy of the Cellular Constituents of Lacticaseibacillus paracasei Zhang in a Normal and Viable but Nonculturable State

by Qiuhua Bao, Xiaoyu Bo, Lu Chen, Yan Ren, Huiying Wang, Lai-Yu Kwok and Wenjun Liu

Microorganisms 2023, 11(5), 1266; https://doi.org/10.3390/microorganisms11051266 - 11 May 2023

Cited by 1 | Viewed by 1604

Abstract

This study aimed to investigate the molecular composition of a viable but nonculturable (VBNC) state of a probiotic strain, Lacticaseibacillus paracasei Zhang (L. paracasei Zhang), using single-cell Raman spectroscopy (SCRS). Fluorescent microcopy with live/dead cell staining (propidium iodide and SYTO 9), plate [...] Read more.

This study aimed to investigate the molecular composition of a viable but nonculturable (VBNC) state of a probiotic strain, Lacticaseibacillus paracasei Zhang (L. paracasei Zhang), using single-cell Raman spectroscopy (SCRS). Fluorescent microcopy with live/dead cell staining (propidium iodide and SYTO 9), plate counting, and scanning electron microscopy were used in combination to observe bacteria in an induced VBNC state. We induced the VBNC state by incubating the cells in de Man, Rogosa, and Sharpe broth (MRS) at 4 °C. Cells were sampled for subsequent analyses before VBNC induction, during it, and up to 220 days afterwards. We found that, after cold incubation for 220 days, the viable plate count was zero, but active cells could still be observed (as green fluorescent cells) under a fluorescence microscope, indicating that Lacticaseibacillus paracasei Zhang entered the VBNC state under these conditions. Scanning electron microscopy revealed the altered ultra-morphology of the VBNC cells, characterized by a shortened cell length and a wrinkled cell surface. Principal component analysis of the Raman spectra profiles revealed obvious differences in the intracellular biochemical constituents between normal and VBNC cells. Comparative analysis of the Raman spectra identified 12 main differential peaks between normal and VBNC cells, corresponding to carbohydrates, lipids, nucleic acids, and proteins. Our results suggested that there were obvious cellular structural intracellular macromolecular differences between normal and VBNC cells. During the induction of the VBNC state, the relative contents of carbohydrates (such as fructose), saturated fatty acids (such as palmitic acid), nucleic acid constituents, and some amino acids changed obviously, which could constitute a bacterial adaptive mechanism against adverse environmental conditions. Our study provides a theoretical basis for revealing the formation mechanism of a VBNC state in lactic acid bacteria. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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12 pages, 2868 KiB

Open AccessArticle

Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening

by Fan Xu, Chuan Liu, Miaomiao Xia, Shixin Li, Ran Tu, Sijia Wang, Hongxing Jin and Dawei Zhang

Microorganisms 2023, 11(4), 1070; https://doi.org/10.3390/microorganisms11041070 - 20 Apr 2023

Cited by 1 | Viewed by 1382

Abstract

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based [...] Read more.

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based microfluidics technology, single cells can be encapsulated in droplets. The screening can be carried out by detecting the fluorescence intensity of secreted riboflavin. Thus, an efficient and high-throughput screening method suitable for riboflavin production strain improvement could be established. In this study, droplet-based microfluidics screening was applied, and a more competitive riboflavin producer U3 was selected from the random mutation library of strain S1. The riboflavin production and biomass of U3 were higher than that of S1 in flask fermentation. In addition, the results of fed-batch fermentation showed that the riboflavin production of U3 was 24.3 g/L, an 18% increase compared with the parent strain S1 (20.6 g/L), and the yield (g riboflavin/100 g glucose) increased by 19%, from 7.3 (S1) to 8.7 (U3). Two mutations of U3 (sinR^G89R and icd^D28E) were identified through whole genome sequencing and comparison. Then they were introduced into BS168DR (parent of S1) for further analysis, which also caused riboflavin production to increase. This paper provides protocols for screening riboflavin-producing B. subtilis with droplet-based microfluidics technology and reveals mutations in riboflavin overproduction strains. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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18 pages, 4226 KiB

Open AccessArticle

Continuous Production of Ethanol, 1-Butanol and 1-Hexanol from CO with a Synthetic Co-Culture of Clostridia Applying a Cascade of Stirred-Tank Bioreactors

by Miriam Bäumler, Veronika Burgmaier, Fabian Herrmann, Julian Mentges, Martina Schneider, Armin Ehrenreich, Wolfgang Liebl and Dirk Weuster-Botz

Microorganisms 2023, 11(4), 1003; https://doi.org/10.3390/microorganisms11041003 - 12 Apr 2023

Cited by 2 | Viewed by 1635

Abstract

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations [...] Read more.

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations and ongoing chain elongation at 800 mbar CO. On/off-gassing with CO indicated a reversible inhibition of C. kluyveri. A continuous supply of sulfide led to increased autotrophic growth and ethanol formation by Clostridium carboxidivorans even at unfavorable low CO concentrations. Based on these results, a continuously operated cascade of two stirred-tank reactors was established with a synthetic co-culture of both Clostridia. An amount of 100 mbar CO and additional sulfide supply enabled growth and chain elongation in the first bioreactor, whereas 800 mbar CO resulted in an efficient reduction of organic acids and de-novo synthesis of C2-C6 alcohols in the second reactor. High alcohol/acid ratios of 4.5–9.1 (w/w) were achieved in the steady state of the cascade process, and the space-time yields of the alcohols produced were improved by factors of 1.9–5.3 compared to a batch process. Further improvement of continuous production of medium chain alcohols from CO may be possible by applying less CO-sensitive chain-elongating bacteria in co-cultures. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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14 pages, 1284 KiB

Open AccessArticle

Magnetite Nanoparticles and Carbon Nanotubes for Improving the Operation of Mesophilic Anaerobic Digesters

by Elvira E. Ziganshina and Ayrat M. Ziganshin

Microorganisms 2023, 11(4), 938; https://doi.org/10.3390/microorganisms11040938 - 03 Apr 2023

Cited by 3 | Viewed by 1408

Abstract

Anaerobic waste processing contributes to the development of the bioenergy sector and solves environmental problems. To date, many technologies have been developed for increasing the rate of the anaerobic digestion process and yield of methane. However, new technological advancements are required to eliminate [...] Read more.

Anaerobic waste processing contributes to the development of the bioenergy sector and solves environmental problems. To date, many technologies have been developed for increasing the rate of the anaerobic digestion process and yield of methane. However, new technological advancements are required to eliminate biogas production inefficiencies. The performance of anaerobic digesters can be improved by adding conductive materials. In this study, the effects of the separate and shared use of magnetite nanoparticles and carbon nanotubes in anaerobic digesters converting high-nitrogen-containing waste, chicken manure, were investigated. The tested nanomaterials accelerated the methane production and increased the decomposition of products from the acidogenesis and acetogenesis stages. The combined use of magnetite nanoparticles and carbon nanotubes gave better results compared to using them alone or without them. Members of the bacterial classes Bacteroidia, Clostridia, and Actinobacteria were detected at higher levels in the anaerobic digesters, but in different proportions depending on the experiment. Representatives of the genera Methanosarcina, Methanobacterium, and Methanothrix were mainly detected within the methanogenic communities in the anaerobic digesters. The present study provides new data for supporting the anaerobic treatment of substrates with a high content of inhibitory compounds, such as chicken wastes. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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14 pages, 2091 KiB

Open AccessArticle

Characterization of Bacillus Strains from Natural Honeybee Products with High Keratinolytic Activity and Antimicrobial Potential

by Diego Martín-González, Sergio Bordel, Selvin Solis, Jorge Gutierrez-Merino and Fernando Santos-Beneit

Microorganisms 2023, 11(2), 456; https://doi.org/10.3390/microorganisms11020456 - 11 Feb 2023

Cited by 3 | Viewed by 1851

Abstract

Two efficient feather-degrading bacteria were isolated from honeybee samples and identified as Bacillus sonorensis and Bacillus licheniformis based on 16S rRNA and genome sequencing. The strains were able to grow on chicken feathers as the sole carbon and nitrogen sources and degraded the [...] Read more.

Two efficient feather-degrading bacteria were isolated from honeybee samples and identified as Bacillus sonorensis and Bacillus licheniformis based on 16S rRNA and genome sequencing. The strains were able to grow on chicken feathers as the sole carbon and nitrogen sources and degraded the feathers in a few days. The highest keratinase activity was detected by the B. licheniformis CG1 strain (3800 U × mL⁻¹), followed by B. sonorensis AB7 (1450 U × mL⁻¹). Keratinase from B. licheniformis CG1 was shown to be active across a wide range of pH, potentially making this strain advantageous for further industrial applications. All isolates displayed antimicrobial activity against Micrococcus luteus; however, only B. licheniformis CG1 was able to inhibit the growth of Mycobacterium smegmatis. In silico analysis using BAGEL and antiSMASH identified gene clusters associated with the synthesis of non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKSs) and/or ribosomally synthesized and post-translationally modified peptides (RiPPs) in most of the Bacillus isolates. B. licheniformis CG1, the only strain that inhibited the growth of the mycobacterial strain, contained sequences with 100% similarity to lichenysin (also present in the other isolates) and lichenicidin (only present in the CG1 strain). Both compounds have been described to display antimicrobial activity against distinct bacteria. In summary, in this work, we have isolated a strain (B. licheniformis CG1) with promising potential for use in different industrial applications, including animal nutrition, leather processing, detergent formulation and feather degradation. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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18 pages, 1767 KiB

Open AccessArticle

Screening Enzymes That Can Depolymerize Commercial Biodegradable Polymers: Heterologous Expression of Fusarium solani Cutinase in Escherichia coli

by Fernando Santos-Beneit, Le Min Chen, Sergio Bordel, Raquel Frutos de la Flor, Octavio García-Depraect, Raquel Lebrero, Sara Rodriguez-Vega, Raúl Muñoz, Rosa Aragão Börner and Tim Börner

Microorganisms 2023, 11(2), 328; https://doi.org/10.3390/microorganisms11020328 - 28 Jan 2023

Cited by 10 | Viewed by 2791

Abstract

In recent years, a number of microbial enzymes capable of degrading plastics have been identified. Biocatalytic depolymerization mediated by enzymes has emerged as a potentially more efficient and environmentally friendly alternative to the currently employed methods for plastic treatment and recycling. However, the [...] Read more.

In recent years, a number of microbial enzymes capable of degrading plastics have been identified. Biocatalytic depolymerization mediated by enzymes has emerged as a potentially more efficient and environmentally friendly alternative to the currently employed methods for plastic treatment and recycling. However, the functional and systematic study of depolymerase enzymes with respect to the degradation of a series of plastic polymers in a single work has not been widely addressed at present. In this study, the ability of a set of enzymes (esterase, arylesterase and cutinase) to degrade commercial biodegradable polymers (PBS, PBAT, PHB, PHBH, PHBV, PCL, PLA and PLA/PCL) and the effect of pre-treatment methods on their degradation rate was assessed. The degradation products were identified and quantified by HPLC and LC-HRMS analysis. Out of the three enzymes, Fusarium solani cutinase (FsCut) showed the highest activity on grinded PBAT, PBS and PCL after 7 days of incubation. FsCut was engineered and heterologous expressed in Escherichia coli, which conferred the bacterium the capability of degrading solid discs of PBAT and to grow in PBS as the sole carbon source of the medium. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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16 pages, 3509 KiB

Open AccessArticle

Accelerated Adaptive Laboratory Evolution by Automated Repeated Batch Processes in Parallelized Bioreactors

by Lukas Bromig and Dirk Weuster-Botz

Microorganisms 2023, 11(2), 275; https://doi.org/10.3390/microorganisms11020275 - 20 Jan 2023

Cited by 3 | Viewed by 2072

Abstract

Adaptive laboratory evolution (ALE) is a valuable complementary tool for modern strain development. Insights from ALE experiments enable the improvement of microbial cell factories regarding the growth rate and substrate utilization, among others. Most ALE experiments are conducted by serial passaging, a method [...] Read more.

Adaptive laboratory evolution (ALE) is a valuable complementary tool for modern strain development. Insights from ALE experiments enable the improvement of microbial cell factories regarding the growth rate and substrate utilization, among others. Most ALE experiments are conducted by serial passaging, a method that involves large amounts of repetitive manual labor and comes with inherent experimental design flaws. The acquisition of meaningful and reliable process data is a burdensome task and is often undervalued and neglected, but also unfeasible in shake flask experiments due to technical limitations. Some of these limitations are alleviated by emerging automated ALE methods on the μL and mL scale. A novel approach to conducting ALE experiments is described that is faster and more efficient than previously used methods. The conventional shake flask approach was translated to a parallelized, L scale stirred-tank bioreactor system that runs controlled, automated, repeated batch processes. The method was validated with a growth optimization experiment of E. coli K-12 MG1655 grown with glycerol minimal media as a benchmark. Off-gas analysis enables the continuous estimation of the biomass concentration and growth rate using a black-box model based on first principles (soft sensor). The proposed method led to the same stable growth rates of E. coli with the non-native carbon source glycerol 9.4 times faster than the traditional manual approach with serial passaging in uncontrolled shake flasks and 3.6 times faster than an automated approach on the mL scale. Furthermore, it is shown that the cumulative number of cell divisions (CCD) alone is not a suitable timescale for measuring and comparing evolutionary progress. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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15 pages, 2471 KiB

Open AccessArticle

CRISPR-Cas9 Mediated Knockout of SagD Gene for Overexpression of Streptokinase in Streptococcus equisimilis

by Armi M. Chaudhari, Sachin Vyas, Vijai Singh, Amrutlal Patel, Chaitanya Joshi and Madhvi N. Joshi

Microorganisms 2022, 10(3), 635; https://doi.org/10.3390/microorganisms10030635 - 17 Mar 2022

Cited by 2 | Viewed by 2728

Abstract

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is higher globally than production due to increased incidences of various heart conditions. The main source [...] Read more.

Streptokinase is an enzyme that can break down the blood clots in some cases of myocardial infarction (heart attack), pulmonary embolism, and arterial thromboembolism. Demand for streptokinase is higher globally than production due to increased incidences of various heart conditions. The main source of streptokinase is various strains of Streptococci. Expression of streptokinase in native strain Streptococcus equisimilis is limited due to the SagD gene-mediated post-translational modification of streptolysin, an inhibitor of streptokinase expression through the degradation of FasX small RNA (through CoV/RS), which stabilizes streptokinase mRNA. In order to improve the stability of mRNA and increase the expression of streptokinase, which is inhibited by SagA, we used CRISPR-Cas9 to successfully knockout the SagD gene and observed a 13.58-fold increased expression of streptokinase at the transcript level and 1.48-fold higher expression at the protein level in the mutant strain compared to wild type. We have demonstrated the successful gene knockout of SagD using CRISPR-Cas9 in S. equisimilis, where an engineered strain can be further used for overexpression of streptokinase for therapeutic applications. Full article

(This article belongs to the Special Issue Advances in Microbial Cell Factories)

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