Bringing a scalable adaptive hybrid modeling framework closer to industrial use: Application on a multiscale fungal fermentation.

Digitalization has paved the way for new paradigms such as digital shadows and digital twins for fermentation processes, opening the door for real-time process monitoring, control, and optimization. With a digital shadow, real-time model adaptation to accommodate complex metabolic phenomena such as metabolic shifts of a process can be monitored. Despite the many benefits of digitalization, the potential has not been fully reached in the industry. This study investigates the development of a digital shadow for a very complex fungal fermentation process in terms of microbial physiology and fermentation operation on pilot-scale at Novonesis and the challenges thereof. The process has historically been difficult to optimize and control due to a lack of offline measurements and an absence of biomass measurements. Pilot-scale and lab-scale fermentations were conducted for model development and validation. With all available pilot-scale data, a data-driven soft sensor was developed to estimate the main substrate concentration (glucose) with a normalized root mean squared error (N-RMSE) of 2%. This robust data-driven soft sensor was able to estimate accurately in lab-scale (volume < 20× pilot) with a N-RMSE of 7.8%. A hybrid soft sensor was developed by combining the data-driven soft sensor with a mass balance to estimate the glycerol and biomass concentrations on pilot-scale data with N-RMSEs of 11% and 21%, respectively. A digital shadow modeling framework was developed by coupling a mechanistic model (MM) with the hybrid soft sensor. The digital shadow modeling framework significantly improved the predictability compared with the MM. The contribution of this study brings the application of digital shadows closer to industrial implementation. It demonstrates the high potential of using this type of modeling framework for scale-up and leads the way to a new generation of in silico-based process development.

Analyzing microbial community and volatile compound profiles in the fermentation of cigar tobacco leaves.

Variations in industrial fermentation techniques have a significant impact on the fermentation of cigar tobacco leaves (CTLs), consequently influencing the aromatic attributes of the resulting cigars. The entire fermentation process of CTLs can be categorized into three distinct phases: phase 1 (CTLs prior to moisture regain), phase 2 (CTLs post-moisture regain and pile fermentation), and phase 3 (CTLs after fermentation and drying). These phases were determined based on the dynamic changes in microbial community diversity. During phase 2, there was a rapid increase in moisture and total acid content, which facilitated the proliferation of Aerococcus, a bacterial genus capable of utilizing reducing sugars, malic acid, and citric acid present in tobacco leaves. In contrast, fungal microorganisms exhibited a relatively stable response to changes in moisture and total acid, with Aspergillus, Alternaria, and Cladosporium being the dominant fungal groups throughout the fermentation stages. Bacterial genera were found to be more closely associated with variations in volatile compounds during fermentation compared to fungal microorganisms. This association ultimately resulted in higher levels of aroma components in CTLs, thereby improving the overall quality of the cigars. These findings reinforce the significance of industrial fermentation in shaping CTL quality and provide valuable insights for future efforts in the artificial regulation of secondary fermentation in CTLs. KEY POINTS: • Industrial fermentation processes impact CTLs microbial communities. • Moisture and total acid content influence microbial community succession in fermentation. • Bacterial microorganisms strongly influence CTLs' aldehyde and ketone flavors over fungi.

Yeast population dynamics in Brazilian bioethanol production.

The large-scale and nonaseptic fermentation of sugarcane feedstocks into fuel ethanol in biorefineries represents a unique ecological niche, in which the yeast Saccharomyces cerevisiae is the predominant organism. Several factors, such as sugarcane variety, process design, and operating and weather conditions, make each of the ∼400 industrial units currently operating in Brazil a unique ecosystem. Here, we track yeast population dynamics in 2 different biorefineries through 2 production seasons (April to November of 2018 and 2019), using a novel statistical framework on a combination of metagenomic and clonal sequencing data. We find that variation from season to season in 1 biorefinery is small compared to the differences between the 2 units. In 1 biorefinery, all lineages present during the entire production period derive from 1 of the starter strains, while in the other, invading lineages took over the population and displaced the starter strain. However, despite the presence of invading lineages and the nonaseptic nature of the process, all yeast clones we isolated are phylogenetically related to other previously sequenced bioethanol yeast strains, indicating a common origin from this industrial niche. Despite the substantial changes observed in yeast populations through time in each biorefinery, key process indicators remained quite stable through both production seasons, suggesting that the process is robust to the details of these population dynamics.

High throughput screening of key functional strains based on improving tobacco quality and mixed fermentation.

Background: Tobacco alcoholization is an important step in increasing the quality of tobacco leaf, which may convert a portion of low-grade tobacco leaves into useable product, however this may take to 2-3 years. The addition of exogenous microorganisms to tobacco leaves and treating them by biological fermentation can shorten the maturation time of tobacco leaves, and improve the quality and applicability of low-grade tobacco leaves Methods: Several strains were screened from low-grade tobacco by flow cytometry, including the bacteria Bacillus amyloliticus, with starch degradation ability and Bacillus kochii, with protein degradation ability, and the fungus Filobasidium magnum with lipid oxidase ability, and were inoculated onto tobacco leaves, both individually and in combination, for solid-state fermentation Results: The greatest improvement in tobacco quality was observed when strains 4# and 3# were applied at a ratio of 3:1. The Maillard reaction products, such as 2-amyl furan, 1-(2-furanmethyl) -1 h-pyrrole, furfural and 2, 5-dimethylpyrazine, were significantly increased, by up to more than 2 times. When strains F7# and 3# were mixed at a ratio of 3:1, the improvement of sensory evaluation index was better than that of pure cultures. The increase of 3-(3, 4-dihydro-2h-pyrro-5-yl) pyridine, ß -damasone and benzyl alcohol was more than 1 times. The increase of 2-amyl-furan was particularly significant, up to 20 times Conclusion: The functional strains screened from tobacco leaves were utilized for the biological fermentation of tobacco leaves, resulting in the reduction of irritation and an improvement in quality of final product, showing a good potential for application.

Challenges to Ensure a Better Translation of Metabolic Engineering for Industrial Applications.

Metabolic engineering has evolved towards creating cell factories with increasingly complex pathways as economic criteria push biotechnology to higher value products to provide a sustainable source of speciality chemicals. Optimization of such pathways often requires high combinatory exploration of best pathway balance, and this has led to increasing use of high-throughput automated strain construction platforms or novel optimization techniques. In addition, the low catalytic efficiency of such pathways has shifted emphasis from gene expression strategies towards novel protein engineering to increase specific activity of the enzymes involved so as to limit the metabolic burden associated with excessively high pressure on ribosomal machinery when using massive overexpression systems. Metabolic burden is now generally recognized as a major hurdle to be overcome with consequences on genetic stability but also on the intensified performance needed industrially to attain the economic targets for successful product launch. Increasing awareness of the need to integrate novel genetic information into specific sites within the genome which not only enhance genetic stability (safe harbors) but also enable maximum expression profiles has led to genome-wide assessment of best integration sites, and bioinformatics will facilitate the identification of most probable landing pads within the genome.To facilitate the transfer of novel biotechnological potential to industrial-scale production, more attention, however, has to be paid to engineering metabolic fitness adapted to the specific stress conditions inherent to large-scale fermentation and the inevitable heterogeneity that will occur due to mass transfer limitations and the resulting deviation away from ideal conditions as seen in laboratory-scale validation of the engineered cells. To ensure smooth and rapid transfer of novel cell lines to industry with an accelerated passage through scale-up, better coordination is required form the onset between the biochemical engineers involved in process technology and the genetic engineers building the new strain so as to have an overall strategy able to maximize innovation at all levels. This should be one of our key objectives when building fermentation-friendly chassis organisms.

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains.

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.

From Sharks to Yeasts: Squalene in the Development of Vaccine Adjuvants.

Squalene is a natural linear triterpene that can be found in high amounts in certain fish liver oils, especially from deep-sea sharks, and to a lesser extent in a wide variety of vegeTable oils. It is currently used for numerous vaccine and drug delivery emulsions due to its stability-enhancing properties and biocompatibility. Squalene-based vaccine adjuvants, such as MF59 (Novartis), AS03 (GlaxoSmithKline Biologicals), or AF03 (Sanofi) are included in seasonal vaccines against influenza viruses and are presently being considered for inclusion in several vaccines against SARS-CoV-2 and future pandemic threats. However, harvesting sharks for this purpose raises serious ecological concerns that the exceptional demand of the pandemic has exacerbated. In this line, the use of plants to obtain phytosqualene has been seen as a more sustainable alternative, yet the lower yields and the need for huge investments in infrastructures and equipment makes this solution economically ineffective. More recently, the enormous advances in the field of synthetic biology provided innovative approaches to make squalene production more sustainable, flexible, and cheaper by using genetically modified microbes to produce pharmaceutical-grade squalene. Here, we review the biological mechanisms by which squalene-based vaccine adjuvants boost the immune response, and further compare the existing sources of squalene and their environmental impact. We propose that genetically engineered microbes are a sustainable alternative to produce squalene at industrial scale, which are likely to become the sole source of pharmaceutical-grade squalene in the foreseeable future.

Genomic Stability and Phenotypic Characteristics of Industrially Produced Lacticaseibacillus rhamnosus GG in a Yogurt Matrix.

Lacticaseibacillus rhamnosus GG is a widely marketed probiotic with well-documented probiotic properties. Previously, deletion of the mucus-adhesive spaCBA-srtC1 genes in dairy isolates was reported. In this study, we examined the genome preservation of industrially produced L. rhamnosus GG (DSM 33156) cofermented in yogurts. In total, DNA of 66 samples, including 60 isolates, was sequenced. Population samples and 59 isolates exhibited an intact genome. One isolate exhibited loss of spaCBA-srtC1. In addition, we examined phenotypes related to the probiotic properties of L. rhamnosus GG either from frozen pellets or cofermented in yogurt. L. rhamnosus GG from frozen pellets induced a response in intestinal barrier function in vitro, in contrast to frozen pellets of the starter culture. Yogurt matrix, containing only the starter culture, induced a response, but cofermentation with L. rhamnosus GG induced a higher response. Conversely, only the starter culture stimulated cytokine secretion in dendritic cells, and it was observed that the addition of L. rhamnosus GG to the starter culture reduced the response. We conclude that the L. rhamnosus GG genome is preserved in yogurt and that common in vitro probiotic effects of L. rhamnosus GG are observed when examined in the yogurt matrix. IMPORTANCELacticaseibacillus rhamnosus GG is a well-documented probiotic strain recognized for its high acid and bile tolerance and properties of adhesion to enterocytes and mucus. The strain exhibits SpaCBA pili, which have been demonstrated to play an important role in adhesion and therefore are relevant for persistence in the gastrointestinal tract. Recently we demonstrated that the genome and phenotypes of L. rhamnosus GG are preserved throughout an industrial production pipeline. However, as gene deletions in L. rhamnosus GG were previously reported for isolates from dairy products, a key question on the genomic stability of L. rhamnosus GG in a yogurt matrix remained. The aim of this study was to analyze genome stability and phenotypic characteristics of L. rhamnosus GG in yogurt. We found that the genome of L. rhamnosus GG is well conserved when the organism is cofermented in yogurt. Some phenotypic characteristics are consistent in all product matrixes, while other characteristics are modulated.

Understanding gradients in industrial bioreactors.

Gradients in industrial bioreactors have attracted substantial research attention since exposure to fluctuating environmental conditions has been shown to lead to changes in the metabolome, transcriptome as well as population heterogeneity in industrially relevant microorganisms. Such changes have also been found to impact key process parameters like the yield on substrate and the productivity. Hence, understanding gradients is important from both the academic and industrial perspectives. In this review the causes of gradients are outlined, along with their impact on microbial physiology. Quantifying the impact of gradients requires a detailed understanding of both fluid flow inside industrial equipment and microbial physiology. This review critically examines approaches used to investigate gradients including large-scale experimental work, computational methods and scale-down approaches. Avenues for future work have been highlighted, particularly the need for further coordinated development of both in silico and experimental tools which can be used to further the current understanding of gradients in industrial equipment.

Primary and Secondary Succession Mediate the Accumulation of Biogenic Amines during Industrial Semidry Chinese Rice Wine Fermentation.

The use of exogenous functional microorganisms to regulate biogenic amine (BA) content is a common approach in fermentation systems. Here, to better understand the microbial traits of succession trajectories in resource-based and biotic interference systems, the BA-related primary and secondary succession were tracked during industrial semidry Chinese rice wine (CRW) fermentation. Dominant abundance and BA-associated microbial functionality based on phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) indicated that Citrobacter, Acinetobacter, Lactobacillus, Exiguobacterium, Bacillus, Pseudomonas, and Enterobacter spp. prominently contributed to the decarboxylase gene family in CRW. The expression levels of tyrosine decarboxylase (tyrDC), ornithine decarboxylase (odc), and agmatine deiminase (aguA) genes were assessed by quantitative PCR (qPCR). The transcription levels of these genes did not correlate with the BA formation rate during postfermentation, indicating that acidification and carbon source depletion upregulated the expression and microbes launch the dormancy strategy to respond to unfavorable conditions. Furthermore, microbial interference with CRW fermentation by Lactobacillus plantarum (ACBC271) and Staphylococcus xylosus (CGMCC1.8382) coinoculated at a ratio of 1:2 exhibited the best synergetic control of BA content. Spearman correlations revealed that Lactobacillus and Staphylococcus exhibited influence on BA-associated microbiota (|ρ| > 0), Exiguobacterium and Pseudomonas were strongly suppressed by Lactobacillus (ρ = -0.867 and ρ = -0.782, respectively; P < 0.05), and Staphylococcus showed the strongest inhibitory effect toward Lactobacillus (ρ = -0.115) and Citrobacter (ρ = -0.188) in the coinoculated 1:2 group. The high inhibitory effect of exogenous added strains on specific bacteria presented evidence for the obtained BA-associated contributors. Overall, this work provides important insight into the microbial traits that rely on resource usage and functional microbiota within food microbial ecology.IMPORTANCE Understanding the shifting patterns of substance usage and microbial interactions is a fundamental objective within microbiology and ecology. Analyses of primary and secondary microbial succession allow for determinations of taxonomic diversity, community traits, and functional transformations over time or after a disturbance. The kinetics of BA generation and the patterns of resource consumption, functional metagenome prediction, and microbial interactions were profiled to elucidate the equilibrium mechanism of microbial systems. Secondary succession after a disturbance triggers a change in resource usage, which in turn affects primary succession and metabolism. In this study, the functional potential of exogenous microorganisms under disturbance synergized with secondary succession strategies, including rebalancing and dormancy, which ultimately reduced BA accumulation. Thus, this succession system could facilitate the settling of essential issues with respect to microbial traits that rely on resource usage and microbial interactions that occur in natural ecosystems.

Exploration of isoxanthohumol bioconversion from spent hops into 8-prenylnaringenin using resting cells of Eubacterium limosum.

Hops is an almost unique source of the potent phytoestrogen 8-prenylnaringenin (8-PN). As hops contain only low levels of 8-PN, synthesis may be more attractive than extraction. A strain of the Gram-positive Eubacterium limosum was isolated previously for 8-PN production from more abundant precursor isoxanthohumol (IX) from hops. In this study, spent hops, an industrial side stream from the beer industry, was identified as interesting source of IX. Yet, hop-derived compounds are well-known antibacterial agents and the traces of a large variety of different compounds in spent hops interfered with growth and IX conversion. Critical factors to finally enable bacterial 8-PN production from spent hops, using a food and feed grade medium, were evaluated in this research. The use of bacterial resting cells and complex medium at a pH of 7.8-8 best fulfilled the requirements for 8-PN production and generated a solid basis for development of an economic process.

Gene regulation of the Lactobacillus vini in response to industrial stress in the fuel ethanol production.

The industrial ethanol fermentation imposes several stresses to microorganisms. However, some bacterial species are well adapted and manage to endure these harmful conditions. Lactobacillus vini is one of the most found bacteria in these environments, indicating the existence of efficient tolerance mechanisms. In view of this premise, the present study aimed to describe the tolerance of L. vini to several stressing agents encounter in industrial environments and the genetic components of the stress response. In general, L. vini showed significant tolerance to stressors commonly found in fuel-ethanol fermentations, and only doses higher than normally reached in processes restrained its growth. The lag phase and the growth rate were the most responsive kinetic parameter affected. Gene expression analysis revealed that uspII gene positively responded to all conditions tested, a typical profile of a general stress response gene. In addition, the results also revealed aspects of regulatory modules of co-expressed genes responding to different stresses, and also the similarities of response mechanism with basis in common cellular damages. Altogether, these data contribute to uncover the factors that could favour L. vini in the industrial fermentation which could be shared with other well adapted species and reports the first stress response genes in this bacterium.

Lactobacillus rhamnosus GG Genomic and Phenotypic Stability in an Industrial Production Process.

Lactobacillus rhamnosus GG is one of the most widely marketed and studied probiotic strains. In L. rhamnosus GG, the spaCBA-srtC1 gene cluster encodes pili, which are important for some of the probiotic properties of the strain. A previous study showed that the DNA sequence of the spaCBA-srtC1 gene cluster was not present in some L. rhamnosus GG variants isolated from liquid dairy products. To examine the stability of the L. rhamnosus GG genome in an industrial production process, we sequenced the genome of samples of L. rhamnosus GG (DSM 33156) collected at specific steps of the industrial production process, including the culture collection stock, intermediate fermentations, and final freeze-dried products. We found that the L. rhamnosus GG genome sequence was unchanged throughout the production process. Consequently, the spaCBA-srtC1 gene locus was intact and fully conserved in all 31 samples examined. In addition, different production batches of L. rhamnosus GG exhibited consistent phenotypes, including the presence of pili in final freeze-dried products, and consistent characteristics in in vitro assays of probiotic properties. Our data show that L. rhamnosus GG is highly stable in this industrial production process.IMPORTANCELactobacillus rhamnosus GG is one of the best-studied probiotic strains. One of the well-characterized features of the strain is the pili encoded by the spaCBA-srtC1 gene cluster. These pili are involved in persistence in the gastrointestinal tract and are important for the probiotic properties of L. rhamnosus GG. Previous studies demonstrated that the L. rhamnosus GG genome can be unstable under certain conditions and can lose the spaCBA-srtC1 gene cluster. Since in vitro studies have shown that the loss of the spaCBA-srtC1 gene cluster decreases certain L. rhamnosus GG probiotic properties, we assessed both the genomic stability and phenotypic properties of L. rhamnosus GG throughout an industrial production process. We found that neither genomic nor phenotypic changes occurred in the samples. Therefore, we demonstrate that L. rhamnosus GG retains the spaCBA-srtC1 cluster and exhibits excellent genomic and phenotypic stability in the specific industrial process examined here.

Industrial-Scale Production and Applications of Bacterial Cellulose.

Bacterial cellulose (BC) is a natural biomaterial synthesized by bacteria. It possesses a unique structure of cellulose nanofiber-weaved three-dimensional reticulated network that endows it excellent mechanical properties, high water holding capability and outstanding suspension stability. It is also characterized with high purity, high degree of crystallinity, great biocompatibility and biodegradability. Due to these advantages, BC has gained great attentions in both academic and industrial areas. This critical review summarizes the up-to-date development of BC production and application from an industrial perspective. Firstly, a fundamental knowledge of BC's biosynthesis, structure and properties is described, and then recent developments in the industrial fermentation of BC are introduced. Subsequently, the latest commercial applications of BC in the areas of food, personal care, household chemicals, biomedicine, textile, composite resin are summarized. Finally, a brief discussion of future development of BC industry is presented at the end.

Effect of extraction time on content, composition and sensory perception of proanthocyanidins in wine-like medium and during industrial fermentation of Cabernet Sauvignon.

BACKGROUND: The research objectives focused on the extraction of grape tannins during extended maceration. Skins and seeds were extracted separately in a wine-like medium. In parallel, the same grapes were fermented in industrial tanks. The content and structural characteristics of extractable proanthocyanidins (PAs) were determined spectrophotometrically and using UHPLC-DAD-MS/MS, respectively. Skin, seed extracts and fermented wines were characterized in chemical and sensorial terms after different extraction durations. RESULTS: The extraction of high molecular-weight PAs (HMWPs) from seeds increased for up to 20 days, whereas low molecular-weight PAs (LMWPs) reached a plateau earlier. The extraction of HMWPs and LMWPs from skins reached a maximum at the first sampling. Sensory evaluation confirmed greater astringency and bitterness of seed extracts with increasing time. Neither seed nor skin extracts differed statistically in terms of the mean degree of polymerization (mDP) and percentage of galloylation (%G) on different extraction days (except for seeds at the first sampling). During industrial maceration, HMWPs and LMWPs increased up to 12.7% alcohol (9 days of maceration); thereafter, the increase was not significant, whereas the mDP, %G and percentage of prodelphinidins did not significantly change after 11.4% alcohol. There were positive correlations with the wine astringency and PA content. CONCLUSION: Looking at both simulated and industrial maceration, it can be concluded that, with a longer maceration time, the increase in HMWP content was more evident than PA structural changes. The increasing content of tannins from seeds played an important role in the greater astringency and bitterness of Cabernet Sauvignon macerated at length. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Harnessing microbial metabolomics for industrial applications.

Metabolome defines a set of metabolites present in a biological sample, which provides an immediate and dynamic recording of microbes in response to genetic and/or environmental perturbations. In recent years, metabolomics in combination with other omics diagnostic tools such as genomics, transcriptomics and proteomics is focused on addressing open biological questions that accelerate our understanding of the system as a whole and boost the use of systems metabolic engineering tools in industrial settings. In this review article, we summarize the applications of metabolomics to industrial microbial fermentations with respect to the bulk production of organic acids, amino acids, enzymes, antibiotics and therapeutic proteins. In addition, future prospects regarding metabolomics-assisted research are provided.

Identification of a Lactic Acid Bacteria to Degrade Biogenic Amines in Chinese Rice Wine and Its Enzymatic Mechanism.

A L. plantarum, CAU 3823, which can degrade 40% of biogenic amines (BAs) content in Chinese rice wine (CRW) at the end of post-fermentation, was selected and characterized in this work. It would be an optimal choice to add 106 cfu/mL of selected strain into the fermentation broth to decrease the BAs while keeping the character and quality of CRW. Nine amine oxidases were identified from the strain and separated using Sephadex column followed by LC-MS/MS analysis. The purified amine oxidase mixture showed a high monoamine oxidase activity of 19.8 U/mg, and more than 40% of BAs could be degraded. The biochemical characters of the amine oxidases were also studied. This work seeks to provide a better solution to degrade BAs in CRW prior to keeping the character and quality of CRW and a better understanding of the degradability of the strain to the BAs.

Grand Research Challenges for Sustainable Industrial Biotechnology.

Future manufacturing will focus on new, improved products as well as on new and enhanced production methods. Recent biotechnological and scientific advances, such as CRISPR/Cas and various omic technologies, pave the way to exciting novel biotechnological research, development, and commercialization of new sustainable products. Rigorous mathematical descriptions of microbial cells and consortia thereof will enable deeper biological understanding and lead to powerful in silico cellular models. Biological engineering, namely model-based design together with synthetic biology, will accelerate the construction of robust and high-performing microorganisms. Using these organisms, and ambitions towards zero-concepts with respect to emissions and excess resources in bioprocess engineering, industrial biotechnology is expected to become highly integrated into sustainable generations of technology systems.

Betaine supplementation improved L-threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression

BACKGROUND: The supplementation of betaine, an osmoprotective compatible solute, in the cultivation media has been widely used to protect bacterial cells. To explore the effects of betaine addition on industrial fermentation, Escherichia coli THRD, an L-threonine producer, was used to examine the production of L-threonine with betaine supplementation and the underlying mechanism through which betaine functions was investigated. RESULTS: Betaine supplementation in the medium of E. coli THRD significantly improved L-threonine fermentation parameters. The transcription of zwf and corresponding enzyme activity of glucose-6-phosphate dehydrogenase were significantly promoted by betaine addition, which contributed to an enhanced expression of zwf that provided more nicotinamide adenine dinucleotide phosphate (NADPH) for L-threonine synthesis. In addition, as a result of the betaine addition, the betaine-stimulated expression of enhanced green fluorescent protein (eGFP) under the zwf promoter within a plasmid-based cassette proved to be a transcription-level response of zwf. Finally, the promoter of the phosphoenolpyruvate carboxylase gene ppc in THRD was replaced with that of zwf, while L-threonine fermentation of the new strain was promoted by betaine addition. Conclusions: We reveal a novel mode of betaine that facilitates the microbial production of useful compounds. Betaine supplementation upregulates the expression of zwf and increases the NADPH synthesis, which may be beneficial for the cell growth and thereby promote the production of L-threonine. This finding might be useful for the production of NADPH-dependent amino acids and derivatives in E. coli THRD or other E. coli strains.

Characterization of a novel lytic bacteriophage from an industrial Escherichia coli fermentation process and elimination of virulence using a heterologous CRISPR-Cas9 system.

Bacterial-bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.