Strains In Co-culture Research Articles

Aminolipids elicit functional trade-offs between competitiveness and bacteriophage attachment in Ruegeria pomeroyi

Lipids play a crucial role in maintaining cell integrity and homeostasis with the surrounding environment. Cosmopolitan marine roseobacter clade (MRC) and SAR11 clade bacteria are unique in that, in addition to glycerophospholipids, they also produce an array of amino acid-containing lipids that are conjugated with beta-hydroxy fatty acids through an amide bond. Two of these aminolipids, the ornithine aminolipid (OL) and the glutamine aminolipid (QL), are synthesized using the O-acetyltransferase OlsA. Here, we demonstrate that OL and QL are present in both the inner and outer membranes of the Gram-negative MRC bacterium Ruegeria pomeroyi DSS-3. In an olsA mutant, loss of these aminolipids is compensated by a concurrent increase in glycerophospholipids. The inability to produce aminolipids caused significant changes in the membrane proteome, with the membrane being less permeable and key nutrient transporters being downregulated while proteins involved in the membrane stress response were upregulated. Indeed, the import of 14C-labelled choline and dimethylsulfoniopropionate, as a proxy for the transport of key marine nutrients across membranes, was significantly impaired in the olsA mutant. Moreover, the olsA mutant was significantly less competitive than the wild type (WT) being unable to compete with the WT strain in co-culture. However, the olsA mutant unable to synthesize these aminolipids is less susceptible to phage attachment. Together, these data reveal a critical role for aminolipids in the ecophysiology of this important clade of marine bacteria and a trade-off between growth and avoidance of bacteriophage attachment.

Effect of refrigerated storage on some physicochemical characteristics of a teff‐based fermented beverage and the viability of the fermenting Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus used

Probiotic beverages made from cereals become interesting in the recent food industries. In this contribution, a fermented teff-based probiotic beverage was produced using the whole grain teff flour and co-culture strains of Lactiplantibacillus plantarum (LPA6) and Lacticaseibacillus rhamnosus GG (LCGG). Then, the effect of 25 days of refrigerated storage on cell viability (LPA6 and LCGG), and contents of sugars, organic acids, and titratable acidity (TA), as well as pH values were examined. Furthermore, pathogenic microorganisms, hygiene indicators, and sensory tests of the beverage were analyzed. Presumptive cell counts of LPA6 and LCGG were observed to decrease throughout refrigerated storage. Glucose, lactic acid, maltose, and acetic acid contents were significantly (p < 0.05) increased over storage time. Also, pH reduction and TA increment were observed in storage time. Examined pathogenic microorganisms and hygiene indicators were not detected in the beverage. Sensory analysis of the beverage after 10 days of refrigerated storage was accepted by the panelists. Novelty Impact Statement Throughout refrigerated storage of teff-based probiotic beverage sugars and organic acids were produced. Sensory attributes of the newly produced teff-based probiotic beverage were accepted by the panelist after 10 days of refrigerated storage. The pH of the teff-based probiotic beverage became more acidic throughout 25 days of refrigerated storage.

Secondary Metabolite Variation and Bioactivities of Two Marine Aspergillus Strains in Static Co-Culture Investigated by Molecular Network Analysis and Multiple Database Mining Based on LC-PDA-MS/MS.

Co-culture is known as an efficient way to explore the metabolic potential of fungal strains for new antibiotics and other therapeutic agents that could counter emerging health issues. To study the effect of co-culture on the secondary metabolites and bioactivities of two marine strains, Aspergillus terreus C23-3 and Aspergillus. unguis DLEP2008001, they were co-cultured in live or inactivated forms successively or simultaneously. The mycelial morphology and high-performance thin layer chromatography (HPTLC) including bioautography of the fermentation extracts were recorded. Furthermore, the agar cup-plate method was used to compare the antimicrobial activity of the extracts. Based on the above, liquid chromatography-photodiode array-tandem mass spectrometry (LC-PDA-MS/MS) together with Global Natural Products Social molecular networking (GNPS) and multiple natural products database mining were used to further analyze their secondary metabolite variations. The comprehensive results showed the following trends: (1) The strain first inoculated will strongly inhibit the growth and metabolism of the latter inoculated one; (2) Autoclaved A. unguis exerted a strong inducing effect on later inoculated A. terreus, while the autoclaved A. terreus showed high stability of its metabolites and still potently suppressed the growth and metabolism of A. unguis; (3) When the two strains are inoculated simultaneously, they both grow and produce metabolites; however, the A. terreus seemed to be more strongly induced by live A. unguis and this inducing effect surpassed that of the autoclaved A. unguis. Under some of the conditions, the extracts showed higher antimicrobial activity than the axenic cultures. Totally, A. unguis was negative in response but potent in stimulating its rival while A. terreus had the opposite effect. Fifteen MS detectable and/or UV active peaks showed different yields in co-cultures vs. the corresponding axenic culture. GNPS analysis assisted by multiple natural products databases mining (PubChem, Dictionary of Natural Products, NPASS, etc.) gave reasonable annotations for some of these peaks, including antimicrobial compounds such as unguisin A, lovastatin, and nidulin. However, some of the peaks were correlated with antagonistic properties and remain as possible novel compounds without mass or UV matching hits from any database. It is intriguing that the two strains both synthesize chemical ‘weapons’ for antagonism, and that these are upregulated when needed in competitive co-culture environment. At the same time, compounds not useful in this antagonistic setting are downregulated in their expression. Some of the natural products produced during antagonism are unknown chlorinated metabolites and deserve further study for their antimicrobial properties. In summary, this study disclosed the different responses of two Aspergillus strains in co-culture, revealed their metabolic variation, and displayed new opportunities for antibiotic discovery.

Studying the effect of oxygen availability and matrix structure on population density and inter-strain interactions of Listeria monocytogenes in different dairy model systems

Due to the ubiquitous character of Listeria monocytogenes multiple strains of the pathogen may end up co-existing in/on the same final products and could potentially cause infection during consumption. Such multiple strain contamination may occur in different stages of the food supply chain. The present study evaluated the effect of oxygen availability and matrix structure on inter-strain interactions of L. monocytogenes that may occur at high population levels in/on different dairy model systems. L. monocytogenes strains C5 and ScottA (4b), 6179 (1/2a) and PL25 (1/2b) were selected as resistant to different antibiotics (enabling selective enumeration of each strain in co-culture) and inoculated (2.0–3.0 log CFU/mL, g or cm2) in Ricotta and Camembert broth (1 dairy product: 2 ¼ Ringer solution) and in/on dairy-based structured media (dairy broth supplemented with 0.6 and 1.4% agar), in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic, hypoxic and anoxic conditions. Every experimental treatment was tested with three biological replicates and two technical repeats (n = 3 × 2). The simultaneously presence of different strains of the pathogen in/on the same substrate did not affect neither the duration of the lag phase nor the growth rate of the co-cultured strains. The observed inter-strain interactions were related with the final population reached/decrease during storage and occurred after the “critical” population density of ca. 6.0 log CFU/mL, g or cm2. The phenomenon was more pronounced in/on Ricotta than in/on Camembert-based substrates, indicating that the composition and the available nutrients of the substrate may affect the interactions that expressed as difference in the final population level between singly and co-cultured strains. Under aerobic and hypoxic conditions, most of the observed interactions were more pronounced in dairy-based broths and were mitigated with the addition of agar. The elimination of oxygen resulted in a prolonged lag time, which lasted at least 5 days and no observed interactions by the end of storage, due to low microbial counts. Investigating inter-strain interactions during growth in/on different substrates, which may have undergone temperature abuse during their transport along the supply chain or during storage in household refrigerators, could assist in explaining the mismatch between clinical and food samples during outbreak investigations.

Development and optimization of a microbial co-culture system for heterologous indigo biosynthesis

BackgroundIndigo is a color molecule with a long history of being used as a textile dye. The conventional production methods are facing increasing economy, sustainability and environmental challenges. Therefore, developing a green synthesis method converting renewable feedstocks to indigo using engineered microbes is of great research and application interest. However, the efficiency of the indigo microbial biosynthesis is still low and needs to be improved by proper metabolic engineering strategies.ResultsIn the present study, we adopted several metabolic engineering strategies to establish an efficient microbial biosynthesis system for converting renewable carbon substrates to indigo. First, a microbial co-culture was developed using two individually engineered E. coli strains to accommodate the indigo biosynthesis pathway, and the balancing of the overall pathway was achieved by manipulating the ratio of co-culture strains harboring different pathway modules. Through carbon source optimization and application of biosensor-assisted cell selection circuit, the indigo production was improved significantly. In addition, the global transcription machinery engineering (gTME) approach was utilized to establish a high-performance co-culture variant to further enhance the indigo production. Through the step-wise modification of the established system, the indigo bioproduction reached 104.3 mg/L, which was 11.4-fold higher than the parental indigo producing strain.ConclusionThis work combines modular co-culture engineering, biosensing, and gTME for addressing the challenges of the indigo biosynthesis, which has not been explored before. The findings of this study confirm the effectiveness of the developed approach and offer a new perspective for efficient indigo bioproduction. More broadly, this innovative approach has the potential for wider application in future studies of other valuable biochemicals’ biosynthesis.

Interaction analyses based on growth parameters of GWAS between Escherichia coli and Staphylococcus aureus

To accurately explore the interaction mechanism between Escherichia coli and Staphylococcus aureus, we designed an ecological experiment to monoculture and co-culture E. coli and S. aureus. We co-cultured 45 strains of E. coli and S. aureus, as well as each species individually to measure growth over 36 h. We implemented a genome wide association study (GWAS) based on growth parameters (λ, R, A and s) to identify significant single nucleotide polymorphisms (SNPs) of the bacteria. Three commonly used growth regression equations, Logistic, Gompertz, and Richards, were used to fit the bacteria growth data of each strain. Then each equation’s Akaike’s information criterion (AIC) value was calculated as a commonly used information criterion. We used the optimal growth equation to estimate the four parameters above for strains in co-culture. By plotting the estimates for each parameter across two strains, we can visualize how growth parameters respond ecologically to environment stimuli. We verified that different genotypes of bacteria had different growth trajectories, although they were the same species. We reported 85 and 52 significant SNPs that were associated with interaction in E. coli and S. aureus, respectively. Many significant genes might play key roles in interaction, such as yjjW, dnaK, aceE, tatD, ftsA, rclR, ftsK, fepA in E. coli, and scdA, trpD, sdrD, SAOUHSC_01219 in S. aureus. Our study illustrated that there were multiple genes working together to affect bacterial interaction, and laid a solid foundation for the later study of more complex inter-bacterial interaction mechanisms.

Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour.

Fermented cocoa beans (Theobroma cacao L.) are a pivotal raw material for chocolate production. A cocktail yeast applied in the cocoa fermentation process can promote the formation of pleasant metabolites. Saccharomyces, Pichia and Hanseniaspora have been widely used in fermentation to improve the final product organoleptic profile, highlighting that fermentation is a critical point for chocolate flavour precursor production. This study aims to evaluate the impact of Pichia kluyveri and Saccharomyces cerevisiae strains as starter cultures on the fermentation for two cocoa hybrids, FA13 and CEPEC2002. During fermentation processes, volatile organic compounds (VOCs) and protein profiles were assessed. Chocolates produced were also assessed regarding the presence of VOCs. Eighty VOCs were identified using gas chromatography coupled to mass spectrometry analysis. Mass spectrometry provided the protein profile evolution during fermentation and showed that the profiles changed with inoculation type (spontaneous versus inoculated fermentation). Chocolate obtained from FA13 inoculated with S. cerevisiae strain contained a greater amount of organics acids, being categorised as sourer than chocolate produced by spontaneous fermentation of FA13. CEPEC2002 inoculated with S. cerevisiae strain in co-culture with P. kluyveri strain generated less sour and sweeter chocolate than spontaneous fermentation only. Chocolates from inoculated assays with starter cultures were more accepted by evaluators, highlighting that P. kluyveri and S. cerevisiae influence the composition of VOCs. Besides, protein profiles also changed throughout fermentation. Further investigation should be conducted to clarify protein degradation dynamics during inoculated fermentations to define which of the microbial cultures positively affect the chocolate sensory characteristics. © 2021 Society of Chemical Industry.

A bottom-up approach towards a bacterial consortium for the biotechnological conversion of chitin to l-lysine

Chitin is an abundant waste product from shrimp and mushroom industries and as such, an appropriate secondary feedstock for biotechnological processes. However, chitin is a crystalline substrate embedded in complex biological matrices, and, therefore, difficult to utilize, requiring an equally complex chitinolytic machinery. Following a bottom-up approach, we here describe the step-wise development of a mutualistic, non-competitive consortium in which a lysine-auxotrophic Escherichia coli substrate converter cleaves the chitin monomer N-acetylglucosamine (GlcNAc) into glucosamine (GlcN) and acetate, but uses only acetate while leaving GlcN for growth of the lysine-secreting Corynebacterium glutamicum producer strain. We first engineered the substrate converter strain for growth on acetate but not GlcN, and the producer strain for growth on GlcN but not acetate. Growth of the two strains in co-culture in the presence of a mixture of GlcN and acetate was stabilized through lysine cross-feeding. Addition of recombinant chitinase to cleave chitin into GlcNAc2, chitin deacetylase to convert GlcNAc2 into GlcN2 and acetate, and glucosaminidase to cleave GlcN2 into GlcN supported growth of the two strains in co-culture in the presence of colloidal chitin as sole carbon source. Substrate converter strains secreting a chitinase or a β-1,4-glucosaminidase degraded chitin to GlcNAc2 or GlcN2 to GlcN, respectively, but required glucose for growth. In contrast, by cleaving GlcNAc into GlcN and acetate, a chitin deacetylase-expressing substrate converter enabled growth of the producer strain in co-culture with GlcNAc as sole carbon source, providing proof-of-principle for a fully integrated co-culture for the biotechnological utilization of chitin.Graphical abstractKey Points• A bacterial consortium was developed to use chitin as feedstock for the bioeconomy.• Substrate converter and producer strain use different chitin hydrolysis products.• Substrate converter and producer strain are mutually dependent on each other.

Evaluation of bioactive glass scaffolds incorporating SrO or ZnO for bone repair: In vitro bioactivity and antibacterial activity.

A series of bioactive glass scaffolds doped with SrO or ZnO (0, 5, and 10 mol%) were synthesized by the foam replica and melting method. The thermodynamic evolution, phase composition, microstructure, ion release, in vitro bioactivity, and oxygen density of the scaffolds were characterized. The proliferation of murine long bone osteocyte Y4 cells was studied by cell culture. The survival rate of the BGs evaluated the antibacterial activity and Escherichia coli strains in co-culture. The results indicated that the process window decreases with the increase of dopants. All the samples have a pore structure size of 200-400 μm. When the scaffolds were immersed in simulated body fluid for 28 days, hydroxyapatite formation was not affected, but the degradation process was retarded. The glass network packing and ionic radii variations of the substitution ions control surface degradation, glass dissolution, and ion release. MTT results revealed that 5Sr-BG had a significant effect on promoting cell proliferation and none of the BGs were cytotoxicity. Sr-BGs and Zn-BGs exhibited significantly inhibited growth against E. coli bacterial strains. Generally, these results showed the 5Sr-BG scaffold with high vitro bioactivity, cell proliferation, and antibacterial property is an important candidate material for bone tissue regeneration and repair.

Co-culture with Synechococcus facilitates growth of Prochlorococcus under ocean acidification conditions.

Anthropogenic CO2 emissions are projected to lower the pH of the ocean 0.3 units by 2100. Previous studies suggested that Prochlorococcus and Synechococcus, the numerically dominant phytoplankton in the oceans, have different responses to elevated CO2 that may result in a dramatic shift in their relative abundances in future oceans. Here we showed that the exponential growth rates of these two genera respond to future CO2 conditions in a manner similar to other cyanobacteria, but Prochlorococcus strains had significantly lower realized growth rates under elevated CO2 regimes due to poor survival after exposure to fresh culture media. Despite this, a Synechococcus strain was unable to outcompete a Prochlorococcus strain in co-culture at elevated CO2 . Under these conditions, Prochlorococcus' poor response to elevated CO2 disappeared, and Prochlorococcus' relative fitness showed negative frequency dependence, with both competitors having significant fitness advantages when initially rare. These experiments suggested that the two strains should be able to coexist indefinitely in co-culture despite sharing nearly identical nutritional requirements. We speculate that negative frequency dependence exists due to reductive Black Queen evolution that has resulted in a passively mutualistic relationship analogous to that connecting Prochlorococcus with the 'helper' heterotrophic microbes in its environment.

CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli.

Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics in high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here, we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct Escherichia coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications.

Isolation and Single Cell Chemical Biological Analysis of Natural Product Producing Cave Microorganisms

Microbial secondary metabolites (natural products) constitute a large fraction of pharmaceuticals, with applications as antibiotics and anti‐cancer therapeutics. However, the discovery of new natural product drug leads is increasingly difficult as isolation studies using traditional methods, and from traditional sources, have resulted in progressively fewer novel compounds discovered. Historically, searching for new sources of biodiversity from unique ecosystems has resulted in increased discovery rates. The cave environment, a previously uninvestigated ecosystem, is a promising terrain to discover bacteria with novel natural products because of its unique conditions, which include low light, relatively constant temperature year‐round, abundant oxygen content, and low nutrient content (oligotrophy). We hypothesize that the oligotrophic environment present in caves forces microorganisms to compete or cooperate with each other, which require the development of natural products as communication, offensive, or defensive chemicals. Non‐Streptomyces actinomycetes are a group of bacteria found in caves that represent good candidates for the isolation of new natural products. We acquire bacterial samples by swabbing rock formations and collecting sediment inside a cave. These samples are diluted and incubated on a number of different agar plating conditions. Individual colonies are then isolated and identified by their 16S rRNA sequences. Isolated actinomycetes are stimulated using a battery of chemical and biological conditions known to elicit secondary metabolism (coculture strains, heavy metals, and antibiotics), and then culture extracts are analyzed for possible cellular activity using multiplexed biological assays against mammalian cells. Specifically, extracts are tested using single cell analysis via a flow cytometry platform termed Multiplexed Activity Metabolomics (MAM), which identifies compounds for isolation by analyzing extracts and metabolomic arrays against multiple cell status markers.Support or Funding InformationNational Cancer Institutes R01 CA226833

Viability of lactic acid bacteria in different components of Ogi with anti diarrhoeagenic E. coli activities

Background: Ogi constitutes a rich source of lactic acid bacteria (LAB) with associated health benefits to humans through antimicrobial activities. However, the high viability of LAB in Ogi and its supernatant (Omidun) is essential. Aims: This study was carried out to assess the viability of LAB in various forms of modified and natural Ogi and the antimicrobial properties of Omidun against diarrhoeagenic E coli. Methods and Material: The viability of LAB was assessed in fermented Ogi slurry and Omidun for one month and also freeze-dried Ogi with and without added bacterial strains for two months. A further 10 days viability study of modified Omidun, refrigerated Omidun, and normal Ogi was performed. The antimicrobial effects of modified Omidun against five selected strains of diarrhoeagenic E. coli (DEC) were evaluated by the co-culture method. Results: Both drying methods significantly affected carotenoids and phenolic compounds. The Ogi slurry had viable LAB only for 10 days after which, there was a succession of fungi and yeast. Omidun showed 2 log10cfu/ml reduction of LAB count each week and the freeze-dried Ogi showed progressive reduction in viability. Refrigerated Omidun has little viable LAB, while higher viability was seen in modified Omidun (≥2 log cfu/ml) than normal Omidun. Modified Omidun intervention led to 2-4 log reduction in diarrhoeagenic E. coli strains and total inactivation of shigella-toxin producing E. coli H66D strain in co-culture. Conclusions: The consumption of Ogi should be within 10 days of milling using modified Omidun. There are practical potentials of consumption of Omidun in destroying E. coli strains implicated in diarrhea. Keywords: Ogi, Omidun, lactic acid bacteria, diarrhoeagenic Escherichia coli strains, Viability.

Developing E. coli-E. coli co-cultures to overcome barriers of heterologous tryptamine biosynthesis

Tryptamine is an alkaloid compound with demonstrated bioactivities and is also a precursor molecule to many important hormones and neurotransmitters. The high efficiency biosynthesis of tryptamine from inexpensive and renewable carbon substrates is of great research and application significance. In the present study, a tryptamine biosynthesis pathway was established in a metabolically engineered E. coli-E. coli co-culture. The upstream and downstream strains of the co-culture were dedicated to tryptophan provision and conversion to tryptamine, respectively. The constructed co-culture was cultivated using either glucose or glycerol as carbon source for de novo production of tryptamine. The manipulation of the co-culture strains’ inoculation ratio was adapted to balance the biosynthetic strengths of the pathway modules for bioproduction optimization. Moreover, a biosensor-assisted cell selection strategy was adapted to improve the pathway intermediate tryptophan provision by the upstream strain, which further enhanced the tryptamine biosynthesis. The resulting biosensor-assisted modular co-culture produced 194 ​mg/L tryptamine with a yield of 0.02 ​g/g glucose using shake flask cultivation. The findings of this work demonstrate that the biosensor-assisted modular co-culture engineering offers a new perspective for conducting microbial biosynthesis.

Establishing microbial co-cultures for 3-hydroxybenzoic acid biosynthesis on glycerol.

Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment-friendly, and cost-effective production of these value-added products without the reliance on petroleum. In this study, rationally designed E.coli-E.coli co-culture systems were established for converting glycerol to 3-hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. The co-culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co-culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with the E.coli mono-culture, the optimized co-culture showed 5.3-fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co-culture engineering for addressing the challenges of aromatic compound biosynthesis.

Balancing the non-linear rosmarinic acid biosynthetic pathway by modular co-culture engineering

Pathway balancing is a critical and common challenge for microbial biosynthesis using metabolic engineering approaches. Non-linear biosynthetic pathways, such as diverging and converging pathways, are particularly difficult for bioproduction optimization, because they require delicate balancing between all interconnected constituent pathway modules. The emergence of modular co-culture engineering offers a new perspective for biosynthetic pathways modularization and balancing, as the biosynthetic capabilities of individual pathway modules can be coordinated by flexible adjustment of the subpopulation ratio of the co-culture strains carrying the designated modules. This study developed microbial co-cultures composed of multiple metabolically engineered E. coli strains for heterologous biosynthesis of complex natural product rosmarinic acid (RA) whose biosynthesis involves a complex diverging-converging pathway. Our results showed that, compared with the conventional mono-culture strategy, the engineered two-strain co-cultures significantly improved the RA production. Further pathway modularization and balancing in the context of three-strain co-cultures resulted in additional production improvement. Moreover, metabolically engineered co-culture strains utilizing different carbon substrates were recruited to improve the three-strain co-culture stability. The optimized co-culture based on these efforts produced 172 mg/L RA, exhibiting 38-fold biosynthesis improvement over the parent strain used in mono-culture biosynthesis. The findings of this work demonstrate the strong potentials of modular co-culture engineering for overcoming the challenges of complex natural product biosynthesis involving non-linear pathways.

Advances in heterologous biosynthesis of plant and fungal natural products by modular co-culture engineering

Heterologous biosynthesis has been long pursued as a viable approach for high efficiency production of natural products with various industrial values. Conventional methods for heterologous biosynthesis use the mono-culture of an engineered microbe for accommodating the whole target biosynthetic pathway to produce the desired product. The emergence of modular co-culture engineering, which divides the pathway between multiple co-culture strains, presents a new perspective to conduct heterologous biosynthesis and improve the bioproduction performance of natural products. This review highlights recent advances in utilizing the modular co-culture engineering approaches to address the challenges of plant and fungal natural product biosynthesis. Potential directions for future research in this promising field are also discussed.

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy

Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.

Heterologous biosynthesis of natural product naringenin by co-culture engineering

Co-culture engineering is an emerging approach for microbial biosynthesis of a variety of biochemicals. In this study, E. coli-E. coli co-cultures were developed for heterologous biosynthesis of the natural product naringenin. The co-cultures were composed of two independent E. coli strains dedicated to functional expression of different portions of the biosynthetic pathway, respectively. The co-culture biosynthesis was optimized by investigating the effect of carbon source, E. coli strain selection, timing of IPTG induction and the inoculation ratio between the co-culture strains. Compared with the mono-culture strategy, the utilization of the designed co-cultures significantly improved the naringenin production, largely due to the reduction of metabolic stress, employment of proper hosts for improving pathway enzyme activities, and flexible adjustment of the relative biosynthetic strength between the co-culture strains. The findings of this study extend the applicability of co-culture engineering in complex natural product biosynthesis.

Effects of 4 Probiotic Strains in Coculture with Traditional Starters on the Flavor Profile of Yogurt.

To study the influence of probiotics on the flavor profile of yogurt, 4 probiotics, including Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei, were cofermented with traditional starters. The changes of bacterial growth, acid contents and volatile compounds of yogurt were investigated during fermentation and refrigerated storage. The strains that exhibited a low growth rate in milk did not significantly affect the bacterial population dynamics, acidity, or organic acid content during fermentation and storage. However, high viability and enhancement of postacidification were clearly observed in the samples that contained strains with a high growth rate in milk, particularly L. casei. A total of 45 volatile compounds, detected in most samples, were identified by headspace solid-phase micro-extraction followed by gas chromatography-mass spectrometry. Among these compounds, ketones and aldehydes were the most abundant. The presence of either L. rhamnosus or L. plantarum did not significantly affect the major volatile compounds, while contributions of L. casei and L. acidophilus were found in the formation of minor volatile metabolites. Electronic nose measurements exhibited a good discrimination of samples that contained different probiotics during refrigerated storage.