Yuheng Lin

Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli

cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered strain is able to produce 1.2g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of up to 1.5g/L MA within 48h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novo biosynthesis of valuable degradation metabolites.

Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin

4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases--a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (~500 mg l⁻¹ in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance.

Regulating malonyl-CoA metabolism via synthetic antisense RNAs for enhanced biosynthesis of natural products.

Malonyl-CoA is the building block for fatty acid biosynthesis and also a precursor to various pharmaceutically and industrially valuable molecules, such as polyketides and biopolymers. However, intracellular malonyl-CoA is usually maintained at low levels, which poses great challenges to efficient microbial production of malonyl-CoA derived molecules. Inactivation of the malonyl-CoA consumption pathway to increase its intracellular availability is not applicable, since it is usually lethal to microorganisms. In this work, we employ synthetic antisense RNAs (asRNAs) to conditionally down-regulate fatty acid biosynthesis and achieve malonyl-CoA enrichment in Escherichia coli. The optimized asRNA constructs with a loop-stem structure exhibit high interference efficiency up to 80%, leading to a 4.5-fold increase in intracellular malonyl-CoA concentration when fabD gene expression is inhibited. Strikingly, this strategy allows the improved production of natural products 4-hydroxycoumarin, resveratrol, and naringenin by 2.53-, 1.70-, and 1.53-fold in E. coli, respectively. In addition, down-regulation of other fab genes including fabH, fabB, and fabF also leads to remarkable increases in 4-hydroxycoumarin production. This study demonstrates a novel strategy to enhance intracellular malonyl-CoA and indicates the effectiveness of asRNA as a powerful tool for use in metabolic engineering.

Combinatorial biosynthesis of plant-specific coumarins in bacteria

Coumarins are plant secondary metabolites that have demonstrated a variety of important therapeutic properties, such as antibacterial, anti-inflammatory, and anti-coagulant effects, as well as anti-cancer and anti-AIDS activities. However, knowledge regarding their biosynthesis is relatively limited even for the simplest coumarin molecule, which serves as the gateway molecule to many pharmaceutically important coumarin derivatives. Here we reported the design and validation of artificial pathways leading to the biosynthesis of plant-specific simple coumarins in bacteria. First, Escherichia coli strains were engineered to convert inexpensive phenylpropanoid acid precursors, 4-coumarate and ferulate to simple coumarins, umbelliferone (4.3 mg/L) and scopoletin (27.8 mg/L), respectively. Furthermore, we assembled the complete artificial pathways in E. coli and achieved de novo biosynthesis of umbelliferone and scopoletin without addition of precursors. This study lays the foundation for microbial production of more diverse coumarin compounds.

Biological Production of Muconic Acid via a Prokaryotic 2,3-Dihydroxybenzoic Acid Decarboxylase

Non-oxidative decarboxylases belong to a unique enzyme family that does not require any cofactors. Here we report the characterization of a 2,3-dihydroxybenzoic acid (2,3-DHBA) decarboxylase (BDC) from Klebsiella pneumoniae and explore its application on the production of muconic acid. The enzyme properties were systematically studied, including the optimal temperature and pH, kinetic parameters, and substrate specificity. On this basis, we designed an artificial pathway for muconic acid production by connecting 2,3-DHBA biosynthesis with its degradation pathway. Over-expression of entCBA and the key enzymes in the shikimate pathway led to the production of 900 mg L(-1) of 2,3-DHBA. Further, expression of the BDC coupled with catechol 1,2-dioxygenase achieved the conversion of 2,3-DHBA into muconic acid. Finally, assembly of the total pathway resulted in the de novo production of muconic acid up to 480 mg L(-1).

Aerobic biosynthesis of hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate reductase

3-Phenylpropionic acid (3PPA) and 3-(4-hydroxyphenyl) propionic acid (HPPA) are important commodity aromatic acids widely used in food, pharmaceutical and chemical industries. Currently, 3PPA and HPPA are mainly manufactured through chemical synthesis, which contains multiple steps involving toxic solvents and catalysts harmful to environment. Therefore, replacement of such existing petroleum-derived approaches with simple and environmentally friendly biological processes is highly desirable for manufacture of these chemicals. Here, for the first time we demonstrated the de novo biosynthesis of 3PPA and HPPA using simple carbon sources in E. coli by extending the cinnamic acids biosynthesis pathways through biological hydrogenation. We first screened 11 2-enoate reductases (ER) from nine microorganisms, leading to efficient conversion of cinnamic acid and p-coumaric acid to 3PPA and HPPA, respectively. Surprisingly, we found a strictly oxygen-sensitive Clostridia ER capable of functioning efficiently in E. coli even under aerobic conditions. On this basis, reconstitution of the full pathways led to the de novo production of 3PPA and HPPA and the accumulation of the intermediates (cinnamic acid and p-coumaric acid) with cell toxicity. To address this problem, different expression strategies were attempted to optimize individual enzyme׳s expression level and minimize intermediates accumulation. Finally, the titers of 3PPA and HPPA reached 366.77mg/L and 225.10mg/L in shake flasks, respectively. This study not only demonstrated the potential of microbial approach as an alternative to chemical process, but also proved the possibility of using oxygen-sensitive enzymes under aerobic conditions.

Establishing a synergetic carbon utilization mechanism for non-catabolic use of glucose in microbial synthesis of trehalose

In nature glucose is a common carbon and energy source for catabolic use and also a building unit of polysaccharides and glycosylated compounds. The presence of strong glucose catabolic pathways in microorganism rapidly decomposes glucose into smaller metabolites and challenges non-catabolic utilization of glucose as C6 building unit or precursor. To address this dilemma, we design a synergetic carbon utilization mechanism (SynCar), in which glucose catabolism is inactivated and a second carbon source (e.g. glycerol) is employed to maintain cell growth and rationally strengthen PEP driving force for glucose uptake and non-catabolic utilization. Remarkably, a trehalose biosynthesis model developed for proof-of-concept indicates that SynCar leads to 131% and 200% improvement in trehalose titer and yield, respectively. The conversion rate of glucose to trehalose reaches 91% of the theoretical maximum. This work demonstrates the broad applicability of SynCar in the biosynthesis of molecules derived from non-catabolic glucose.

Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters and amides

Caffeic acid has been widely recognized as a versatile pharmacophore for synthesis of new chemical entities, among which caffeic acid derived phenethyl esters and amides are the most extensively-investigated bioactive compounds with potential therapeutical applications. However, the natural biosynthetic routes for caffeic acid derived phenethyl esters or amides remain enigmatic, limiting their bio-based production. Herein, product-directed design of biosynthetic schemes allowed the development of thermodynamically favorable pathways for these compounds via acyltransferase (ATF) mediated trans-esterification. Production based screening identified a microbial O-ATF from Saccharomyces cerevisiae and a plant N-ATF from Capsicum annuum capable of forming caffeic acid derived esters and amides, respectively. Subsequent combinatorial incorporation of caffeic acid with various aromatic alcohol or amine biosynthetic pathways permitted the de novo bacterial production of a panel of caffeic acid derived phenethyl esters or amides in Escherichia coli for the first time. Particularly, host strain engineering via systematic knocking out endogenous caffeoyl-CoA degrading thioesterase and pathway optimization via titrating co-substrates enabled production enhancement of five caffeic acid derived phenethyl esters and amides, with titers ranging from 9.2 to 369.1mg/L. This platform expanded the capabilities of bacterial production of high-value natural aromatic esters and amides from renewable carbon source via tailoring non-natural biosynthetic pathways.

Elevating 4-Hydroxycoumarin Production through Alleviating Thioesterase-mediated Salicoyl-CoA Degradation

Acyl-CoAs are essential intermediates in the biosynthetic pathways of a number of industrially and pharmaceutically important molecules. When these pathways are reconstituted in a heterologous microbial host for metabolic engineering purposes, the acyl-CoAs may be subject to undesirable hydrolysis by the hosts native thioesterases, resulting in a waste of cellular energy and decreased intermediate availability, thus impairing bioconversion efficiency. 4-hydroxycoumarin (4HC) is a direct synthetic precursor to the commonly used oral anticoagulants (e.g. warfarin) and rodenticides. In our previous study, we have established an artificial pathway for 4HC biosynthesis in Escherichia coli, which involves the thioester intermediate salicoyl-CoA. Here, we utilized the 4HC pathway as a demonstration to examine the negative effect of salicoyl-CoA degradaton, identify and inactivate the responsible thioesterase, and eventually improve the 4HC production. We screened a total of 16 E. coli thioesterases and tested their hydrolytic activity towards salicoyl-CoA in vitro. Among all the tested candidate enzymes, YdiI was found to be the dominant contributor to the salicoyl-CoA degradation in E. coli. Remarkably, the ydiI knockout strain carrying the 4HC pathway exhibited an up to 300% increase in 4HC production. An optimized 4HC pathway construct introduced in the ydiI knockout strain led to the accumulation of 935mg/L of 4HC in shake flasks, which is about 1.5 folds higher than the wild-type strain. This study demonstrates a systematic strategy to alleviate the undesirable hydrolysis of thioester intermediates, allowing production enhancement for other biosynthetic pathways with similar issues.

Sensor-regulator and RNAi based bifunctional dynamic control network for engineered microbial synthesis

Writing artificial logic and dynamic function into complex cellular background to achieve desired phenotypes or improved outputs calls for the development of new genetic tools as well as their innovative use. In this study, we present a sensor-regulator and RNAi-based bifunctional dynamic control network that can provide simultaneous upregulation and downregulation of cellular metabolism for engineered biosynthesis. The promoter-regulator-mediated upregulation function and its transduced downregulation function through RNAi are systematically verified and characterized. We apply this dynamic control network to regulate the phosphoenolpyruvate metabolic node in Escherichia coli and achieve autonomous distribution of carbon flux between its native metabolism and the engineered muconic acid biosynthetic pathway. This allows muconic acid biosynthesis to reach 1.8 g L−1. This study also suggests the circumstances where dynamic control approaches are likely to take effects.Engineering dynamic control can improve microbial production of target chemicals. Here, the authors design a sensor-regulator and RNAi based bifunctional dynamic control network that can simultaneously and independently turn up and down cellular metabolism for engineered muconic acid production in E. coli.