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.

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.

[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.

Application and population control strategy of microbial modular co-culture engineering / 生物工程学报

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.

BACKGROUND: Indigo 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. RESULTS: In 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. CONCLUSION: This 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.

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 20.3 mg 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 29.7 mg/L sakuranetin from 5 g/L glucose within 48 h, 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 79.0 mg/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.

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 â€‹mg/L tryptamine with a yield of 0.02 â€‹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.

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.

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.

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.

Modular co-culture engineering, a new approach for metabolic engineering.

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed.