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The objective of this work was to determine in vitro probiotic activity traits of 11 lactic acid bacteria (LAB) strains isolated from pulque obtained from three different locations in the Mexican states of Oaxaca and Puebla using the probiotic strain Lactobacillus acidophilus NCFM as a positive control, and to detect their production of antimicrobial peptides, including bacteriocins and peptidoglycan hydrolases (PGH). The LAB isolates were identified by sequencing of their 16S rRNA as belonging to four different genera of the Lactobacillaceae family: Lactiplantibacillus, Levilactobacillus, Lacticaseibacillus and Liquorilactobacillus, corresponding to the species plantarum, brevis, paracasei and ghanensis, respectively. Most of the strains showed resistance to high acidity (pH 2) and bile salts (0.5%), with survival rates up to 87 and 92%, respectively. In addition, most of the strains presented good antimicrobial activity against the foodborne pathogens Listeria monocytogenes, ECEC and Salmonella Typhi. The strain Liquorilactobacillus ghanensis RVG6, newly reported in pulque, presented an outstanding overall performance on the probiotic activity tests. In terms of their probiotic activity traits assessed in this work, the strains compared positively with the control L. acidophilus NCFM, which is a very-well documented probiotic strain. For the antimicrobial peptide studies, four strains presented bacteriocin-like mediated antibiosis and six had significant PGH activity, with two strains presenting outstanding overall antimicrobial peptide production: Lacticaseibacillus paracasei RVG3 and Levilactobacillus brevis UTMB2. The probiotic performance of the isolates was mainly dependent on strain specificity. The results obtained in this work can foster the revalorization of pulque as a functional natural product. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00684-7.

Adaptive laboratory evolution (ALE) has been used to study and solve pressing questions about evolution, especially for the study of the development of mutations that confer increased fitness during evolutionary processes. In this contribution, we investigated how the evolutionary process conducted with the PTS- mutant of Escherichia coli PB11 in three parallel batch cultures allowed the restoration of rapid growth with glucose as the carbon source. The significant findings showed that genomic sequence analysis of a set of newly evolved mutants isolated from ALE experiments 2-3 developed some essential mutations, which efficiently improved the fast-growing phenotypes throughout different fitness landscapes. Regulator galR was the target of several mutations such as SNPs, partial and total deletions, and insertion of an IS1 element and thus indicated the relevance of a null mutation of this gene in the adaptation of the evolving population of PB11 during the parallel ALE experiments. These mutations resulted in the selection of MglB and GalP as the primary glucose transporters by the evolving population, but further selection of at least a second adaptive mutation was also necessary. We found that mutations in the yfeO, rppH, and rng genes improved the fitness advantage of evolving PTS- mutants and resulted in amplification of leaky activity in Glk for glucose phosphorylation and upregulation of glycolytic and other growth-related genes. Notably, we determined that these mutations appeared and were fixed in the evolving populations between 48 and 72 h of cultivation, which resulted in the selection of fast-growing mutants during one ALE experiments in batch cultures of 80 h duration.Key points• ALE experiments selected evolved mutants through different fitness landscapes in which galR was the target of different mutations: SNPs, deletions, and insertion of IS.• Key mutations in evolving mutants appeared and fixed at 48-72 h of cultivation.• ALE experiments led to increased understanding of the genetics of cellular adaptation to carbon source limitation.

Pulqueis a traditional Mexican fermented, non-distilled alcoholic beverage produced by fermenting the fresh sap (aguamiel) extracted from severalAgave(maguey) species cultivated for pulque production (mainlyA. salmiana). This beverage was produced and consumed since Pre-Hispanic times by Mesoamerican civilizations, mainly in the Mexican Central Plateau, and is one of the essential alcoholic beverages produced and consumed during several centuries in Mexico. By 2019, annual pulque production was reported in 171,482 billion liters. Nevertheless, traditional pulque production faces several significant limitations, including the disappearance of large agave plantations and the extent of time required (at least 5 years) to complete the plant maturation for aguamiel extraction; traditional production practices; and the lack of an efficient stabilization process of the fermented product resulting in low shelf life. In opposition, successful examples of sustainable cultivation of maguey species for aguamiel extraction and the fermentation process's industrialization resulted in high-quality pulque production, with international exportation certification. In this contribution, we present a review of the most relevant aspects of the history and commercial relevance of pulque, the causes that resulted in its production debacle during the first half of the twentieth century, the current situation of its traditional production, and the successful efforts of industrial production of the beverage. We describe recent results on the analysis of the physicochemical characteristics of aguamiel and on the microbiology of the beverage explored by metagenomic techniques that can be proposed as a baseline to redefine the quality criteria of the beverage and to define for the first time a microbiological core to optimize its production. We describe the relevance of maguey species for aguamiel production as a fundamental element of agroforestry and the relevance of its sustainable production, in four sustainable plantation models to maintain a stable plant population to ensure the continuous extraction of aguamiel and pulque production. Finally, we describe some successful examples of beverage industrialization and potential applications of several microorganisms isolated from aguamiel, pulque, aguamiel concentrates, and the maguey to produce high-value bioactive products.

The aminoshikimic acid (ASA) pathway comprises a series of reactions resulting in the synthesis of 3-amino-5-hydroxybenzoic acid (AHBA), present in bacteria such as Amycolatopsis mediterranei and Streptomyces. AHBA is the precursor for synthesizing the mC7N units, the characteristic structural component of ansamycins and mitomycins antibiotics, compounds with important antimicrobial and anticancer activities. Furthermore, aminoshikimic acid, another relevant intermediate of the ASA pathway, is an attractive candidate for a precursor for oseltamivir phosphate synthesis, the most potent anti-influenza neuraminidase inhibitor treatment of both seasonal and pandemic influenza. This review discusses the relevance of the key intermediate AHBA as a scaffold molecule to synthesize diverse ansamycins and mitomycins. We describe the structure and control of the expression of the model biosynthetic cluster rif in A. mediterranei to synthesize ansamycins and review several current pharmaceutical applications of these molecules. Additionally, we discuss some relevant strategies developed for overproducing these chemicals, focusing on the relevance of the ASA pathway intermediates kanosamine, AHAB, and ASA.

Pulque is a culturally important 4,000-year-old traditional Mexican fermented drink. Pulque is produced by adding fresh aguamiel (agave sap) to mature pulque, resulting in a mixture of microbial communities and chemical compositions. We performed shotgun metagenomic sequencing of five stages of pulque fermentation to characterize organismal and functional diversity. We identified 6 genera (Acinetobacter, Lactobacillus, Lactococcus, Leuconostoc, Saccharomyces and Zymomonas) and 10 species (Acinetobacter boissieri, Acinetobacter nectaris, Lactobacillus sanfranciscensis, Lactococcus lactis, Lactococcus piscium, Lactococcus plantarum, Leuconostoc citreum, Leuconostoc gelidum, Zymomonas mobilis and Saccharomyces cerevisiae) that were present ≥ 1% in at least one stage of pulque fermentation. The abundance of genera and species changed during fermentation and was associated with a decrease in sucrose and increases in ethanol and lactic acid, suggesting that resource competition shapes organismal diversity. We also predicted functional profiles, based on organismal gene content, for each fermentation stage and identified an abundance of genes associated with the biosynthesis of folate, an essential B-vitamin. Additionally, we investigated the evolutionary relationships of S. cerevisiae and Z. mobilis, two of the major microbial species found in pulque. For S. cerevisiae, we used a metagenomics assembly approach to identify S. cerevisiae scaffolds from pulque, and performed phylogenetic analysis of these sequences along with a collection of 158 S. cerevisiae strains. This analysis suggests that S. cerevisiae from pulque is most closely related to Asian strains isolated from sake and bioethanol. Lastly, we isolated and sequenced the whole-genomes of three strains of Z. mobilis from pulque and compared their relationship to seven previously sequenced isolates. Our results suggest pulque strains may represent a distinct lineage of Z. mobilis.

Pseudomonas chlororaphis is a plant-associated bacterium with reported antagonistic activity against different organisms and plant growth–promoting properties. P. chlororaphis possesses exciting biotechnological features shared with another Pseudomonas with a nonpathogenic phenotype. Part of the antagonistic role of P. chlororaphis is due to its production of a wide variety of phenazines. To expand the knowledge of the metabolic traits of this organism, we constructed the first experimentally validated genome-scale model of P. chlororaphis ATCC 9446, containing 1267 genes and 2289 reactions, and analyzed strategies to maximize its potential for the production of phenazine-1-carboxamide (PCN). The resulting model also describes the capability of P. chlororaphis to carry out the denitrification process and its ability to consume sucrose (Scr), trehalose, mannose, and galactose as carbon sources. Additionally, metabolic network analysis suggested fatty acids as the best carbon source for PCN production. Moreover, the optimization of PCN production was performed with glucose and glycerol. The optimal PCN production phenotype requires an increased carbon flux in TCA and glutamine synthesis. Our simulations highlight the intrinsic H2O2 flux associated with PCN production, which may generate cellular stress in an overproducing strain. These results suggest that an improved antioxidative strategy could lead to optimal performance of phenazine-producing strains of P. chlororaphis., This work was supported by Dirección General de Asuntos del Personal Académico- Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (DGAPA-PAPIIT), Universidad Nacional Autónoma de México, grant IN211420.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS− derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH−) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH− mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS− and rppH− mutations in the deep of a proteomic approach to understanding the relevance of rppH− phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH−, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH− were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS− strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS− derivatives during the ALE experiment. This result suggests that the evolving PTS− population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH− mutation in the PTS− background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

The fast-growing capability of Escherichia coli strains used to produce industrially relevant metabolites relies on their capability to transport efficiently glucose or potential industrial feedstocks such as sucrose or xylose as carbon sources. E. coli imports extracellular glucose into the periplasmic space across the outer membrane porins: OmpC, OmpF, and LamB. As the internal membrane is an impermeable barrier for sugars, the cell employs several primary and secondary active transport systems, and the phosphoenolpyruvate (PEP)-sugar phosphotransferase (PTS) system for glucose transport. PTS:glucose is the preferred system by E. coli to transport and phosphorylate the periplasmic glucose; nevertheless, PTS imposes a strict metabolic control mechanism on the preferential consumption of glucose over other carbon sources in sugar mixtures such as glucose and xylose resulting from the hydrolysis of lignocellulosic biomass, by the carbon catabolite repression. In this contribution, we summarize the major sugar transport systems for glucose and disaccharide transport, the exhibited substrate plasticity, and their impact on the growth of E. coli, highlighting the relevance of PTS in the control of the expression of genes for the transport and catabolism of other sugars as xylose. We discuss the strategies developed by evolved mutants of E. coli during adaptive laboratory evolution experiments to overcome the nutritional stress condition imposed by inactivation of PTS as a strategy for the selection of fast-growing derivatives in glucose, xylose, or mixtures of glucose:xylose. This approach results in the recruitment of other primary and secondary active transporters, demonstrating relevant sugar plasticity in derivative-evolved mutants. Elucidation of the molecular and biochemical basis of sugar-transport substrate plasticity represents a consistent approach for sugar-transport system engineering for the design of efficient E. coli derivative strains with improved substrate assimilation for biotechnological purposes.

The culture of engineered Escherichia coli for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, ydiB) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of E. coli for SA production remains unclear. We report the effect of the inactivation or the overexpression of ydiB in E. coli strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of ydiB resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of ydiB caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative ydiB strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC50 588 and 589 µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.

Laboratory and industrial cultures of Escherichia coli employ media containing glucose which is mainly transported and phosphorylated by the phosphotransferase system (PTS). In these strains, 50% of the phosphoenolpyruvate (PEP), which results from the catabolism of transported glucose, is used as a phosphate donor for its phosphorylation and translocation by the PTS. This characteristic of the PTS limits the production of industrial biocommodities that have PEP as a precursor. Furthermore, when E. coli is exposed to carbohydrate mixtures, the PTS prevents expression of catabolic and non-PTS transport genes by carbon catabolite repression and inducer exclusion. In this contribution, we discuss the main strategies developed to overcome these potentially limiting effects in production strains. These strategies include adaptive laboratory evolution selection of PTS- Glc+ mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source. We discuss the benefits of using alternative glucose transport systems and describe the application of these strategies to E. coli strains with specific genetic modifications in target pathways. These efforts have resulted in significant improvements in the production of diverse biocommodities, including aromatic metabolites, biofuels and organic acids.

Saponins have been correlated with the reduction of cancer cell growth and the apoptotic effect of agave sap concentrate. Empirical observations of this artisanal Mexican food have shown that fermentation occurs after agave sap is concentrated, but little is known about the microorganisms that survive after cooking, or their effects on saponins and other metabolites. The aim of this study was to evaluate the changes in metabolites found in agave (A. salmiana) sap after its fermentation with microorganisms isolated from agave sap concentrate, and demonstrate its potential use to enhance anticancer activity. Microorganisms were isolated by dilution plating and identified by 16S rRNA analysis. Isolates were used to ferment agave sap, and their corresponding butanolic extracts were compared with those that enhanced the cytotoxic activity on colon (Caco-2) and liver (Hep-G2) cancer cells. Metabolite changes were investigated by mass spectrometry-based metabolomics. Among 69 isolated microorganisms, the actinomycetes Arthrobacter globiformis and Gordonia sp. were used to analyze the metabolites, along with bioactivity changes. From the 939 ions that were mainly responsible for variation among fermented samples at 48 h, 96 h, and 192 h, four were correlated to anticancer activity. It was shown that magueyoside B, a kammogenin glycoside, was found at higher intensities in the samples fermented with Gordonia sp. that reduced Hep-G2 viability better than controls. These findings showed that microorganisms from agave sap concentrate change agave sap metabolites such as saponins. Butanolic extracts obtained after agave sap fermentation with Arthrobacter globiformis or Gordonia sp. increased the cancer cell growth inhibitory effect on colon or liver cancer cells, respectively.

Pseudomonas chlororaphis strain ATCC 9446 is a biocontrol-related organism. We report here its draft genome sequence assembled into 35 contigs consisting of 6,783,030 bp. Genome annotation predicted a total of 6,200 genes, 6,128 coding sequences, 81 pseudogenes, 58 tRNAs, 4 noncoding RNAs (ncRNAs), and 41 frameshifted genes.

Pseudomonas chlororaphis strain ATCC 9446 is a biocontrol-related organism. We report here its draft genome sequence assembled into 35 contigs consisting of 6,783,030 bp. Genome annotation predicted a total of 6,200 genes, 6,128 coding sequences, 81 pseudogenes, 58 tRNAs, 4 noncoding RNAs (ncRNAs), and 41 frameshifted genes.

Pulque is the fermented product of aguamiel that has been consumed in Mexico since pre-Hispanic times. Aguamiel is the sap obtained from agave plants about 8–10 years old after their immature flower stem is removed. Due to the aguamiel extraction process and the natural fermentation, pulque is the product of the metabolism of a complex microbiota. Besides alcohol, the concentration and complexity of polysaccharides is one of the main characteristics of pulque. Along with the phytochemicals naturally found in aguamiel, its microbiota composition is affected by the environmental conditions and the collection time. In general, this product is still obtained by a very artisanal process and, therefore, its potential health benefits have been difficult to evaluate. It is an excellent source of prebiotics and probiotics but its consumption must be limited due to the alcohol content.

a b s t r a c t In the present work we describe an enzymatic production method to obtain 2-6 fructose oligosaccha- rides (levan-type FOS) through a sequential reaction in which a bacterial endolevanase is applied to levan produced from sucrose by bacterial levansucrases. A putative gene encoding an endolevanase, designated as LevBl, was identified through a bioinformatics search, isolated from a strain of Bacillus licheniformis IBt1 from our own collection and expressed in Escherichia coli. LevB1 showed a specific activity of 1.8 U/mg protein at 35 ◦C in 50 mM phosphate buffer pH 6.0. A first order kinetic behavior was found when up to 150 g/L of low molecular weight levan (8.3 kDa) was used as the substrate. The product profile was determined by HPAEC-PAD and consisted of levan-type FOS with a polymerization degree between 2 and 8, with levanbiose as the major product after long reaction times. Yields of 97% of levan-type FOS were obtained when 1.0 U/mL of LevB1 reacted with 100 g/L of levan produced by the levansucrase from Bacillus subtilis. Finally, it was observed that levan-type FOS are efficiently fermented by probiotic lactic acid bacteria. © 2014 Elsevier Ltd. All rights reserved.

The glycolytic intermediate phosphoenolpyruvate (PEP) is a precursor of several cellular components, including various aromatic compounds. Modifications to the PEP node such as PEP:sugar phosphotransferase system (PTS) or pyruvate kinase inactivation have been shown to have a positive effect on aromatics production capacity in Escherichia coli and Bacillus subtilis. In this study, pyruvate kinase and PTS-deficient B. subtilis strains were employed for the construction of derivatives lacking shikimate kinase activity that accumulate two industrially valuable chemicals, the intermediates of the common aromatic pathway, shikimic and dehydroshikimic acids. The pyruvate kinase-deficient strain (CLC6-PYKA) showed the best production parameters under resting-cell conditions. Compared to the PTS-deficient strain, the shikimic and dehydroshikimic acids specific production rates for CLC6-PYKA were 1.8- and 1.7-fold higher, respectively. A batch fermentor culture using complex media supplemented with 83 g/l of glucose was developed with strain CLC6-PYKA, where final titers of 4.67 g/l (shikimic acid) and 6.2 g/l (dehydroshikimic acid) were produced after 42 h.

Pulque is a traditional Mexican alcoholic beverage produced from the fermentation of the fresh sap known as aguamiel (mead) extracted from several species of Agave (maguey) plants that grow in the Central Mexico plateau. Currently, pulque is produced, sold and consumed in popular districts of Mexico City and rural areas. The fermented product is a milky white, viscous, and slightly acidic liquid beverage with an alcohol content between 4 and 7° GL and history of consumption that dates back to pre-Hispanic times. In this contribution, we review the traditional pulque production process, including the microbiota involved in the biochemical changes that take place during aguamiel fermentation. We discuss the historical relevance and the benefits of pulque consumption, its chemical and nutritional properties, including the health benefits associated with diverse lactic acid bacteria with probiotic potential isolated from the beverage. Finally, we describe the actual status of pulque production as well as the social, scientific and technological challenges faced to preserve and improve the production of this ancestral beverage and Mexican cultural heritage.

The culture of engineered Escherichia coli for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, ydiB) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of E. coli for SA production remains unclear. We report the effect of the inactivation or the overexpression of ydiB in E. coli strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of ydiB resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of ydiB caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative ydiB strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.

Aims: This work describes the isolation and characterization of two new alkaliphilic micro-organisms present in nejayote. Methods and Results: Samples of fresh industrial nejayote were plated on nejayote medium and incubated for 4 days at 37°C. Isolates were identified based on morphological and physiological characteristics, as well as 16S rDNA sequence analysis. Two gram-positive strains, NJY2 and NJY4, able to hydrolyse starch, xylan, and gelatin were isolated from nejayote. Comparative sequence analysis of 16S rDNA and phylogenetic studies indicate that the micro-organisms studied were closely related to members of the Bacillus flexus species. The strains were identified as facultative alkaliphilic salt tolerant bacteria. Isolate NJY2 produced cell associated phenolic acid esterases, able to release ferulic acid from nixtamalised corn bran and ethyl and methyl esters. Conclusions: The isolated strains of B. flexus NJY2 and NJY4 showed important physiological properties to produce high-value molecules from agroindustrial by-products. Significance and Impact of the Study: This is the first report about the isolation of alkaliphilic micro-organisms from nejayote and the first report of phenolic acid esterases synthesised by alkaliphiles. The new alkaliphilic micro-organisms have potential application in the treatment and transformation of tortilla industry residues.

In this study, the characterization of the bacterial community present during the fermentation of pulque, a traditional Mexican alcoholic beverage from maguey (Agave), was determined for the first time by a polyphasic approach in which both culture and non-culture dependent methods were utilized. The work included the isolation of lactic acid bacteria (LAB), aerobic mesophiles, and 16S rDNA clone libraries from total DNA extracted from the maguey sap (aguamiel) used as substrate, after inoculation with a sample of previously produced pulque and followed by 6-h fermentation. Microbiological diversity results were correlated with fermentation process parameters such as sucrose, glucose, fructose and fermentation product concentrations. In addition, medium rheological behavior analysis and scanning electron microscopy in aguamiel and during pulque fermentation were also performed. Our results showed that both culture and non-culture dependent approaches allowed the detection of several new and previously reported species within the alpha-, gamma-Proteobacteria and Firmicutes. Bacteria diversity in aguamiel was composed by the heterofermentative Leuconostoc citreum, L. mesenteroides, L. kimchi, the gamma-Proteobacteria Erwinia rhapontici, Enterobacter spp. and Acinetobacter radioresistens. Inoculation with previously fermented pulque incorporated to the system microbiota, homofermentative lactobacilli related to Lactobacillus acidophilus, several alpha-Proteobacteria such as Zymomonas mobilis and Acetobacter malorum, other gamma-Proteobacteria and an important amount of yeasts, creating a starting metabolic diversity composed by homofermentative and heterofermentative LAB, acetic and ethanol producing microorganisms. At the end of the fermentation process, the bacterial diversity was mainly composed by the homofermentative Lactobacillus acidophilus, the heterofermentative L. mesenteroides, Lactococcus lactis subsp. lactis and the alpha-Proteobacteria A. malorum. After a 6-h fermentation, 83.27% of total sugars detected after inoculation were consumed (228.4 mM hexose equivalents) and a carbon (C) recovery of 66.18% in fermentation products was estimated. They were produced 284.4 mM C as ethanol, 71.5 mM C as acetic acid and 19 mM C as lactic acid, demonstrating the presence of homo- and heterofermentative, acetic and alcoholic metabolisms in the final product. It was also found, after hydrolysis, that the exopolysaccharide produced during the fermentation was mainly composed by fructose residues, probably inulin or levan.

The capacity to utilize sucrose as a carbon and energy source (Scr+ phenotype) is a highly variable trait among Escherichia coli strains. In this study, seven enteropathogenic E. coli (EPEC) strains from different sources were studied for their capacity to grow using sucrose. Liquid media cultures showed that all analyzed strains have the Scr+ phenotype and two distinct groups were defined: one of five and another of two strains displaying doubling times of 67 and 125 min, respectively. The genes conferring the Scr+ phenotype in one of the fast-growing strains (T19) were cloned and sequenced. Comparative sequence analysis revealed that this strain possesses the scr regulon genes scrKYABR, encoding phosphoenolpyruvate:phosphotransferase system-dependent sucrose transport and utilization activities. Transcript level quantification revealed sucrose-dependent induction of scrK and scrR genes in fast-growing strains, whereas no transcripts were detected in slow-growing strains. Sequence comparison analysis revealed that the scr genes in strain T19 are almost identical to those present in the scr regulon of prototype EPEC E2348/69 and in both strains, the scr genes are inserted in the chromosomal intergenic region of hypothetical genes ygcE and ygcF. Comparison of the ygcE-ygcF intergenic region sequence of strains MG1655, enterohemorrhagic EDL933, uropathogenic ECFT073 and EPEC T19-E2348/69 revealed that the number of extragenic highly repeated iap sequences corresponded to nine, four, two and none, respectively. These results show that the iap sequence-containing chromosomal ygcE-ygcF intergenic region is highly variable in E. coli.

In Escherichia coli the phosphotransferase system (PTS) consumes one molecule of phosphoenolpyruvate (PEP) to phosphorylate each molecule of internalized glucose. PEP bioavailability into the aromatic pathway can be increased by inactivating the PTS. However, the lack of the PTS results in decreased glucose transport and growth rates. To overcome such drawbacks in a PTS– strain and reconstitute rapid growth on glucose phenotype (Glc+), the glk and galP genes were cloned into a plasmid and the arcA gene was inactivated. Simultaneous overexpression of glk and galP increased the growth rate and regenerated a Glc+ phenotype. However, the highest growth rate was obtained when glk and galP were overexpressed in the arcA– background. These results indicated that the arcA mutation enhanced glycolytic and respiratory capacities of the engineered strain.

Pulque is a Mexican traditional alcoholic, non-distilled, fermented beverage produced by the fermentation of the sap, known as aguamiel, extracted from several maguey (Agave) species. Pulque has traditionally been considered a healthy beverage due to its nutrient content and also a traditional medicine for the treatment of gastrointestinal disorders and intestinal infections. During pulque fermentation, the development of acidity, alcohol and viscosity define its final sensorial properties, developing an enriched environment where dominant lactic acid bacteria (LAB), including diverse Leuconostoc species, are present. Because traditional pulque is consumed directly from the fermentation vessel, the naturally associated LAB are ingested and reach the human small intestine alive. Here, we report the in vitro and in vivo probiotic assessment of Leuconostoc mesenteroides strain P45 isolated from pulque. This isolated LAB species exhibited lysozyme, acid (pH 3.5) and bile salts (0.1 and 0.3 % oxgall) resistance. Antibacterial activity against the pathogens Listeria monocytogenes, enteropathogenic Escherichia coli, Salmonella enterica serovar Typhi and S. enterica serovar Typhimurium were observed in assays involving cell-to-cell contact, cell-free 2× concentrated supernatants and cell-to-cell contact under exopolysaccharide-producing conditions. The in vivo probiotic assessment showed an anti-infective activity of L. mesenteroides P45 against S. enterica serovar Typhimurium in challenged male and female BALB/c mice. Analysis of the available genome sequence of strain P45 allowed identified a pre-bacteriocin coding gene and six peptidoglycan hydrolase enzymes, probably involved in the antimicrobial activity of this strain. The results presented in this study support some potential microbial mechanisms associated with the beneficial effects on human health of this LAB involved in the fermentation of pulque. Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-2370-7) contains supplementary material, which is available to authorized users.

Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several “intuitive” metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.

Background As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (µ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. As result, several evolved strains increased their growth fitness on glucose as the only carbon source. Two of these clones isolated at 120 and 200 h during the experiment, increased their μ by 338 and 373 %, respectively, compared to the predecessor PB11 strain. The genome sequence and analysis of the genetic changes of these two strains (PB12 and PB13) allowed for the identification of a novel strategy to enhance carbon utilization to overcome the absence of the major glucose transport system. Results Genome sequencing data of evolved strains revealed the deletion of chromosomal region of 10,328 pb and two punctual non-synonymous mutations in the dhaM and glpT genes, which occurred prior to their divergence during the early stages of the evolutionary process. Deleted genes related to increased fitness in the evolved strains are rppH, aas, lplT and galR. Furthermore, the loss of mutH, which was also lost during the deletion event, caused a 200-fold increase in the mutation rate. Conclusions During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the μ of these derivatives. Finally, we demonstrated the deletion of the aas-lplT operon, which codes for the main components of the phosphatidylethanolamine turnover metabolism increased the further fitness and glucose uptake in these evolved strains by stimulating the phospholipid degradation pathway. This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions. The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0382-6) contains supplementary material, which is available to authorized users.

Laboratory and industrial cultures of Escherichia coli employ media containing glucose which is mainly transported and phosphorylated by the phosphotransferase system (PTS). In these strains, 50% of the phosphoenolpyruvate (PEP), which results from the catabolism of transported glucose, is used as a phosphate donor for its phosphorylation and translocation by the PTS. This characteristic of the PTS limits the production of industrial biocommodities that have PEP as a precursor. Furthermore, when E. coli is exposed to carbohydrate mixtures, the PTS prevents expression of catabolic and non-PTS transport genes by carbon catabolite repression and inducer exclusion. In this contribution, we discuss the main strategies developed to overcome these potentially limiting effects in production strains. These strategies include adaptive laboratory evolution selection of PTS(-) Glc(+) mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source. We discuss the benefits of using alternative glucose transport systems and describe the application of these strategies to E. coli strains with specific genetic modifications in target pathways. These efforts have resulted in significant improvements in the production of diverse biocommodities, including aromatic metabolites, biofuels and organic acids.

Phosphoenolpyruvate (PEP) is a key intermediate of cellular metabolism and a precursor of commercially relevant products. In Escherichia coli 50% of the glucose-derived PEP is consumed by the PEP:carbohydrate phosphotransferase system (PTS) for glucose transport. PTS, encoded by the ptsHIcrr operon, was deleted from JM101 to generate strain PB11 (PTS-Glc-). PB12, a mutant derived from PB11, grows faster than the parental strain on glucose (PTS-Glc+ phenotype). This strain can redirect some of the PEP not utilized by PTS into the high yield synthesis of aromatic compounds from glucose. Here, we report a comparative transcription analysis among these strains of more than 100 genes involved in central carbon metabolism during growth on glucose. It was found that in the PTS- strains that have reduced glucose transport capacities, several genes encoding proteins with functions related to carbon transport and metabolism were upregulated. Therefore, it could be inferred that these strains synthesize autoinducers of these genes when sensing very low internal glucose concentrations, probably for scavenging purposes. This condition that is permanently present in the PTS- strains even when growing in high glucose concentrations allowed the simultaneous utilization of glucose and acetate as carbon sources. It was found that the gal operon is upregulated in these strains, as well as the aceBAK, poxB and acs genes among others. In PB12, glk, pgi, the TCA cycle and certain respiratory genes are also upregulated. A mutation in arcB in PB12 is apparently responsible for the upregulation of the TCA cycle and certain respiratory genes.

The physiological role of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been studied in Escherichia coli. It has been shown that it directly or indirectly regulates the activity of most catabolic genes involved in carbohydrate transport. Accordingly, strains lacking PTS have pleiotropic phenotypes and are impaired in their capacity to grow on glucose and other PTS sugars. We have previously reported the characterization of a mutant harboring a pts operon deletion (PB11) which, as expected, showed a severe reduction of its growth capacity when incubated on glucose as carbon source, as compared to that of the isogenic wild-type strain. These observations corroborate that PTS is the main determinant of the capacity to grow on glucose and confirm the existence of other systems that allow glucose utilization although at a reduced level. To explore the physiological state and the metabolic pathways involved in glucose utilization in a pts– background, we analyzed the global transcriptional response of the PB11 mutant when growing in minimal medium with glucose as carbon source. Genome-wide transcriptional analysis using microarrays revealed that, under this condition, expression of several genes related to carbon transport and metabolism was upregulated, as well as that of genes encoding transporters for certain nucleotides, nitrogen, phosphorus and sulfur sources. In addition, upregulation of rpoS and several genes transcribed by this sigma subunit was detected. These results indicate that the reduced capacity of glucose utilization present in the PB11 strain induces a general nutrient-scavenging response and this behavior is not dependent on a functional PTS. This condition is responsible of the utilization of secondary carbon sources in the presence of glucose.

We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (µ) in glucose minimal medium of 0.1 h–1 is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a µ of 0.70 h–1). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a µ of 0.40 h–1. In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB–derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS– strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.

Leuconostoc mesenteroides P45 was isolated from the traditional Mexican pulque beverage. We report its draft genome sequence, assembled in 6 contigs consisting of 1,874,188 bp and no plasmids. Genome annotation predicted a total of 1,800 genes, 1,687 coding sequences, 52 pseudogenes, 9 rRNAs, 51 tRNAs, 1 noncoding RNA, and 44 frameshifted genes.

Agave tequilana Weber var. 'Azul' is grown for the production of tequila, inulin and syrup. Diverse bacteria inhabit plant tissues and play a crucial role for plant health and growth. In this study culturable endophytic bacteria were extracted from leaf bases of 100 healthy Agave tequilana plants. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant tissue. Three hundred endophytic strains were isolated and 16s rDNA sequences grouped the bacteria into eight different taxa that shared high homology with other known sequences. Bacterial endophytes were identified as Acinectobacter sp., A. baumanii, A. bereziniae, Cronobacter sakazakii, Enterobacter hormaechei, Bacillus sp. Klebsiella oxytoca, Pseudomonas sp., Enterococcus casseliflavus, Leuconostoc mesenteroides subsp. mesenteroides and Gluconobacter oxydans. Isolates were confirmed to be plant growth promoting bacteria (PGPB) by their capacities for nitrogen fixation, auxin production, phosphate solubilization, or antagonism against Fusarium oxysporum AC132. E. casseliflavus JM47 and K. oxytoca JM26 secreted the highest concentrations of IAA. The endophyte Acinectobacter sp. JM58 exhibited the maximum values for nitrogen fixation and phosphate solubilization index (PSI). Inhibition of fungi was found in Pseudomonas sp. JM9p and K. oxytoca JM26. Bacterial endophytes show promise for use as bio-inoculants for agave cultivation. Use of endophytes to enhance cultivation of agave may be particularly important for plants produced by micropropagation techniques, where native endophytes may have been lost.

We report the screening and characterization of EPS produced by LAB identified as Leuconostoc kimchii isolated from pulque, a traditional Mexican fermented, non-distilled alcoholic beverage produced by the fermentation of the sap extracted from several (Agave) maguey species. EPS-producing LAB constitutes an abundant bacterial group relative to total LAB present in sap and during fermentation, however, only two EPS-producing colony phenotypes (EPSA and EPSB, respectively) were detected and isolated concluding that despite the high number of polymer-producing LAB their phenotypic diversity is low. Scanning electron microcopy analysis during EPS-producing conditions revealed that both types of EPS form a uniform porous structure surrounding the bacterial cells. The structural characterization of the soluble and cell-associated EPS fractions of each polymer by enzymatic and acid hydrolysis, as by 1D- and 2D-NMR, showed that polymers produced by the soluble and cell-associated fractions of EPSA strain are dextrans consisting of a linear backbone of linked α-(1→6) Glcp in the main chain with α-(1→2) and α-(1→3)-linked branches. The polymer produced by the soluble fraction of EPSB strain was identified as a class 1 dextran with a linear backbone containing consecutive α-(1→6)-linked D-glucopyranosyl units with few α-(1→3)-linked branches, whereas the cell-associated EPS is a polymer mixture consisting of a levan composed of linear chains of (2→6)-linked β-D-fructofuranosyl residues with β-(2→6) connections, and a class 1 dextran. According to our knowledge this is the first report of dextrans and a levan including their structural characterization produced by L. kimchii isolated from a traditional fermented source.

The production of aromatic amino acids using fermentation processes with recombinant microorganisms can be an advantageous approach to reach their global demands. In addition, a large array of compounds with alimentary and pharmaceutical applications can potentially be synthesized from intermediates of this metabolic pathway. However, contrary to other amino acids and primary metabolites, the artificial channelling of building blocks from central metabolism towards the aromatic amino acid pathway is complicated to achieve in an efficient manner. The length and complex regulation of this pathway have progressively called for the employment of more integral approaches, promoting the merge of complementary tools and techniques in order to surpass metabolic and regulatory bottlenecks. As a result, relevant insights on the subject have been obtained during the last years, especially with genetically modified strains of Escherichia coli. By combining metabolic engineering strategies with developments in synthetic biology, systems biology and bioprocess engineering, notable advances were achieved regarding the generation, characterization and optimization of E. coli strains for the overproduction of aromatic amino acids, some of their precursors and related compounds. In this paper we review and compare recent successful reports dealing with the modification of metabolic traits to attain these objectives. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0126-z) contains supplementary material, which is available to authorized users.

Dulce Catalina Díaz Quiroz, Susy Beatriz Carmona, Francisco Bolívar, Adelfo Escalante Department of Cellular Engineering and Biocatalysis, Institute of Biotechnology, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México Abstract: Aromatic metabolism comprises the shikimic acid (SA) and the aminoshikimic acid (ASA) pathways. The SA pathway is the common route for the biosynthesis of aromatic amino acids and other metabolites in bacteria, higher plants, fungi, and Apicomplexa parasites, but this pathway is absent in mammals. A variant of the SA pathway known as the ASA pathway branches off from the normal pathway in some bacteria, and its final product, 3-amino-5-hydroxybenzoic acid, is the precursor for many aminoglycoside antibiotics such as kanamycin, neomycin, butirosin, and spectinomycin. The SA pathway includes the key intermediate SA, which is the precursor for the chemical synthesis of the drug oseltamivir phosphate, known commercially as Tamiflu®, an efficient inhibitor of the neuraminidase enzyme of the seasonal influenza viruses types A and B, avian influenza virus H5N1, and human influenza virus H1N1. Meanwhile, the intermediate of the ASA pathway, ASA, is an attractive candidate for use as the core scaffold for the synthesis of combinatorial libraries and is a potential alternative to SA as a precursor for oseltamivir phosphate synthesis. In this review, we discuss the relevance of the key intermediates SA and ASA as scaffold molecules for the synthesis of diverse chemicals. We highlight the current and potential pharmaceutical applications of these molecules and discuss the main strategies for the production of these aromatic compounds from natural sources and the application of metabolic engineering strategies in diverse bacterial strains for production through biotechnological processes. Keywords: aromatic metabolism, shikimic acid, aminoshikimic acid, oseltamivir phosphate, medical applications, metabolic engineering

The lactic acid bacteria diversity of pozol, a Mexican fermented maize dough, was studied using a total DNA extraction and purification procedure and PCR amplification of 16S rDNA for gram-positive and related bacterial groups. Thirty-six clones were obtained and sequenced to 650 nucleotides. These partial sequences were identified by submission to the non-redundant nucleotide database of NCBI. The identified sequences were aligned with reference sequences of the closest related organisms. This analysis indicated that only 14 sequences were unique clones and these were identified as Lactococcus lactis, Streptococcus suis, Lactobacillus plantarum, Lact. casei, Lact. alimentarium, and Lact. delbruekii and Clostridium sp. Two non-ribosomal sequences were also detected. Unlike other environments analyzed with this molecular approach where many unidentified microorganisms are found, the identity of most sequences could be established as lactic acid bacteria, indicating that this is the main group among the gram-positive bacteria in pozol. Use of this molecular method permitted detection of lactic acid bacteria different from those previously isolated and identified by culture techniques

Background Efficient production of SA in Escherichia coli has been achieved by modifying key genes of the central carbon metabolism and SA pathway, resulting in overproducing strains grown in batch- or fed-batch-fermentor cultures using a complex broth including glucose and YE. In this study, we performed a GTA to identify those genes significantly upregulated in an engineered E. coli strain, PB12.SA22, in mid EXP (5 h), early STA (STA1, 9 h), and late STA (STA2, 44 h) phases, grown in complex fermentation broth in batch-fermentor cultures. Results Growth of E. coli PB12.SA22 in complex fermentation broth for SA production resulted in an EXP growth during the first 9 h of cultivation depending of supernatant available aromatic amino acids provided by YE because, when tryptophan was totally consumed, cells entered into a second, low-growth phase (even in the presence of glucose) until 26 h of cultivation. At this point, glucose was completely consumed but SA production continued until the end of the fermentation (50 h) achieving the highest accumulation (7.63 g/L of SA). GTA between EXP/STA1, EXP/STA2 and STA1/STA2 comparisons showed no significant differences in the regulation of genes encoding enzymes of central carbon metabolism as in SA pathway, but those genes encoding enzymes involved in sugar, amino acid, nucleotide/nucleoside, iron and sulfur transport; amino acid catabolism and biosynthesis; nucleotide/nucleoside salvage; acid stress response and modification of IM and OM were upregulated between comparisons. Conclusions GTA during SA production in batch-fermentor cultures of strain PB12.SA22 grown in complex fermentation broth during the EXP, STA1 and STA2 phases was studied. Significantly, upregulated genes during the EXP and STA1 phases were associated with transport, amino acid catabolism, biosynthesis, and nucleotide/nucleoside salvage. In STA2, upregulation of genes encoding transporters and enzymes involved in the synthesis and catabolism of Arg suggests that this amino acid could have a key role in the fuelling of carbon toward SA synthesis, whereas upregulation of genes involved in pH stress response, such as membrane modifications, suggests a possible response to environmental conditions imposed on the cell at the end of the fermentation.