Modular Co-culture Engineering Research Articles

Structural diversification of bioactive bibenzyls through modular co-culture leading to the discovery of a novel neuroprotective agent.

Bibenzyls, a kind of important plant polyphenols, have attracted growing attention for their broad and remarkable pharmacological activities. However, due to the low abundance in nature, uncontrollable and environmentally unfriendly chemical synthesis processes, these compounds are not readily accessible. Herein, one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes. Three types of efficiently post-modifying modular strains were engineered by employing methyltransferases, prenyltransferase, and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules. Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes. Especially, a prenylated bibenzyl derivative (12) was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke. RNA-seq, quantitative RT-PCR, and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor, mitochondria associated 3 (Aifm3), suggesting that Aifm3 might be a new target in ischemic stroke therapy. This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.

Modular co-culture engineering of Yarrowia lipolytica for amorphadiene biosynthesis

Amorphadiene is the precursor to synthesize the antimalarial drug artemisinin. The production of amorphadiene and artemisinin from metabolically engineered microbes may provide an alternate to plant secondary metabolite extraction. Microbial consortia can offer division of labor, and microbial co-culture system can be leveraged to achieve cost-efficient production of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene (encoding amorphadiene synthase) into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum (ER) surface area, we were able to significantly improve amorphadiene production in this work. Using Po1g/PPtM and Po1f/AaADSERx3/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer were increased to 65.094 mg/L. The enlargement of the ER surface area caused by the deletion of the PAH1 gene provided more subcellular ER space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that the importance of the spatial localization of critical enzymes, and manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of isoprenoid-related secondary metabolites in a modular manner.Graphical

Application and population control strategy of microbial modular co-culture engineering

Traditional methods of microbial synthesis usually rely on a single engineered strain to synthesize the target product through metabolic engineering. The key cofactors, precursors and energy are produced by the introduced complex synthetic pathways. This would increase the physiological burden of engineering strains, resulting in a decrease in the yield of target products. The modular co-culture engineering has become an attractive solution for effective heterologous biosynthesis, where product yield can be greatly improved. In the modular co-culture engineering, the coordination between the population of different modules is essential for increasing the production efficiency. This article summarized recent advances in the application of modular co-culture engineering and population control strategies.

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.

Notable Improvement of 3-Hydroxypropionic Acid and 1,3-Propanediol Coproduction Using Modular Coculture Engineering and Pathway Rebalancing

Sustainable bioconversion of glycerol into 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) is often bottlenecked by the accumulation of a toxic metabolic intermediate, 3-hydroxypropana...

Constructing E. coli Co-Cultures for De Novo Biosynthesis of Natural Product Acacetin.

Modular co-culture engineering is an emerging approach for biosynthesis of complex natural products. In this study, microbial co-cultures composed of two and three Escherichia coli strains, respectively, are constructed for de novo biosynthesis of flavonoid acacetin, a value-added natural compound possessing numerous demonstrated biological activities, from simple carbon substrate glucose. To this end, the heterologous biosynthetic pathway is divided into different modules, each of which is accommodated in a dedicated E. coli strain for functional expression. After the optimization of the inoculation ratio between the constituent strains, the engineered co-cultures show a 4.83-fold improvement in production comparing to the mono-culture controls. Importantly, cultivation of the three-strain co-culture in shake flasks result in the production of 20.3mg L-1 acacetin after 48 h. To the authors' knowledge, this is the first report on acacetin de novo biosynthesis in a heterologous microbial host. The results of this work confirm the effectiveness of modular co-culture engineering for complex flavonoid biosynthesis.

De novo biosynthesis of complex natural product sakuranetin using modular co-culture engineering.

Flavonoids are a large family of plant and fungal natural products, among which many have been found to possess outstanding biological activities. Utilization of engineered microbes as surrogate hosts for heterologous biosynthesis of flavonoids has been investigated extensively. However, current microbial biosynthesis strategies mostly rely on using one microbial strain to accommodate the long and complicated flavonoid pathways, which presents a major challenge for production optimization. Here, we adapt the emerging modular co-culture engineering approach to rationally design, establish and optimize an Escherichia coli co-culture for de novo biosynthesis of flavonoid sakuranetin from simple carbon substrate glucose. Specifically, two E. coli strains were employed to accommodate the sakuranetin biosynthesis pathway. The upstream strain was engineered for pathway intermediate p-coumaric acid production, whereas the downstream strain converted p-coumaric acid to sakuranetin. Through step-wise optimization of the co-culture system, we were able to produce 29.7mg/L sakuranetin from 5g/L glucose within 48h, which is significantly higher than the production by the conventional monoculture-based approach. The co-culture biosynthesis was successfully scaled up in a fed-batch bioreactor, resulting in the production of 79.0mg/L sakuranetin. To our knowledge, this is the highest bioproduction concentration reported so far for de novo sakuranetin biosynthesis using the heterologous host E. coli. The findings of this work expand the applicability of modular co-culture engineering for addressing the challenges associated with heterologous biosynthesis of complex natural products. KEY POINTS: • De novo biosynthesis of sakuranetin was achieved using E. coli-E. coli co-cultures. • Sakuranetin production by co-cultures was significantly higher than the mono-culture controls. • The co-culture system was optimized by multiple metabolic engineering strategies. • The co-culture biosynthesis was scaled up in fed-batch bioreactor.

A Combinatorial Approach to Optimize the Production of Curcuminoids From Tyrosine in Escherichia coli.

Curcuminoids are well-known for their therapeutic properties. However, their extraction from natural sources is environmentally unfriendly, expensive and limited by seasonal variability, highlighting the need for alternative production processes. We propose an optimized artificial biosynthetic pathway to produce curcuminoids, including curcumin, in Escherichia coli. This pathway involves six enzymes, tyrosine ammonia lyase (TAL), 4-coumarate 3-hydroxylase (C3H), caffeic acid O-methyltransferase (COMT), 4-coumarate-CoA ligase (4CL), diketide-CoA synthase (DCS), and curcumin synthase (CURS1). Curcuminoids pathway was divided in two modules, the first module included TAL, C3H and COMT and the second one 4CL, DCS and CURS1. Optimizing the first module of the pathway, from tyrosine to ferulic acid, enabled obtaining the highest ferulic acid titer reported so far (1325.1 μM). Afterward, ferulic acid was used as substrate to optimize the second module of the pathway. We achieved the highest concentration of curcumin ever reported (1529.5 μM), corresponding to a 59.4% increase. Subsequently, curcumin and other curcuminoids were produced from tyrosine (using the whole pathway) in mono-culture. The production increased comparing to a previously reported pathway that used a caffeoyl-CoA O-methyltransferase enzyme (to convert caffeoyl-CoA to feruloyl-CoA) instead of COMT (to convert caffeic to ferulic acid). Additionally, the potential of a co-culture approach was evaluated to further improve curcuminoids production by reducing cells metabolic burden. We used one E. coli strain able to convert tyrosine to ferulic acid and another able to convert the hydroxycinnamic acids produced by the first one to curcuminoids. The co-culture strategies tested led to 6.6 times increase of total curcuminoids (125.8 μM) when compared to the mono-culture system. The curcuminoids production achieved in this study corresponds to a 6817% improvement. In addition, by using an inoculation ratio of 2:1, although total curcuminoids production decreased, curcumin production was enhanced and reached 43.2 μM, corresponding to an improvement of 160% comparing to mono-culture system. To our knowledge, these values correspond to the highest titers of curcuminoids obtained to date. These results demonstrate the enormous potential of modular co-culture engineering to produce curcumin, and other curcuminoids, from tyrosine.

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.

De novo phenol bioproduction from glucose using biosensor-assisted microbial coculture engineering.

Microbial biosynthesis has been extensively adapted for the production of commodity chemicals using renewable feedstocks. This study integrated metabolite biosensors into rationally designed microbial cocultures to achieve high-efficiency bioproduction of phenol from simple carbon substrate glucose. Specifically, two sets of E. coli-E. coli cocultures were first constructed for accommodation of two independent phenol biosynthesis pathways via 4-hydroxybenzoate (4HB) and tyrosine (TYR), respectively. Biosensor-assisted microbial cell selection mechanisms were subsequently incorporated into the coculture systems to address the insufficient pathway intermediate provision that limited the overall bioproduction. For the 4HB- and TYR-dependent pathways, this approach improved the phenol production by 2.3- and 3.9-fold, respectively, compared to the monoculture controls. Notably, the use of biosensor-assisted cell selection strategy in monocultures resulted in reduced phenol production, highlighting the advantage of coculture engineering for coupling with biosensing. After stepwise optimization, the phenol bioproduction yield of the engineered coculture's reached 0.057 g/g glucose. Furthermore, the coculture biosynthesis was successfully scaled up at both shake flask and bioreactor levels. Overall, the findings of this study demonstrate the outstanding potential of coupling biosensing and modular coculture engineering for advancing microbial biosynthesis of valuable molecules from renewable carbon substrates.

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.

Enhancing Production of Pinene in Escherichia coli by Using a Combination of Tolerance, Evolution, and Modular Co-culture Engineering

α-Pinene is a natural and active monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals, and biofuels. Although it has been successfully produced by genetically engineered microorganisms, the production level of pinene is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. We first improved pinene tolerance to 2.0% and pinene production by adaptive laboratory evolution after atmospheric and room temperature plasma (ARTP) mutagenesis and overexpression of the efflux pump to obtain the pinene tolerant strain Escherichia coli YZFP, which is resistant to fosmidomycin. Through error-prone PCR and DNA shuffling, we isolated an Abies grandis geranyl pyrophosphate synthase variant that outperformed the wild-type enzyme. To balance the expression of multiple genes, a tunable intergenic region (TIGR) was inserted between A. grandis GPPSD90G/L175P and Pinus taeda Pt1Q457L. In an effort to improve the production, an E. coli-E. coli modular co-culture system was engineered to modularize the heterologous mevalonate (MEV) pathway and the TIGR-mediated gene cluster of A. grandis GPPSD90G/L175P and P. taeda Pt1Q457L. Specifically, the MEV pathway and the TIGR-mediated gene cluster were integrated into the chromosome of the pinene tolerance strain E. coli YZFP and then evolved to a higher gene copy number by chemically induced chromosomal evolution, respectively. The best E. coli-E. coli co-culture system of fermentation was found to improve pinene production by 1.9-fold compared to the mono-culture approach. The E. coli-E. coli modular co-culture system of whole-cell biocatalysis further improved pinene production to 166.5 mg/L.