Xinxiao Sun

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.

A novel muconic acid biosynthesis approach by shunting tryptophan biosynthesis via anthranilate.

ABSTRACT Muconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid.

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).

Systematically Engineering Escherichia coli for Enhanced Production of 1,2-Propanediol and 1-Propanol

The biological production of high value commodity 1,2-propanediol has been established by engineering the glycolysis pathway. However, the simultaneous achievement of high titer and high yield has not been reported yet, as all efforts in increasing the titer have resulted in low yields. In this work, we overcome this limitation by employing an optimal minimal set of enzymes, channeling the carbon flux into the 1,2-propanediol pathway, increasing NADH availability, and improving the anaerobic growth of the engineered Escherichia coli strain by developing a cell adaptation method. These efforts lead to 1,2-propanediol production at a titer of 5.13 g/L with a yield of 0.48 g/g glucose in 20 mL shake flask studies. On this basis, we pursue the enhancement of 1-propanol production from the 1,2-propanediol platform. By constructing a fusion diol dehydratase and developing a dual strain process, we achieve a 1-propanol titer of 2.91 g/L in 20 mL shake flask studies. To summarize, we report the production of 1,2-propanediol at enhanced titer and enhanced yield simultaneously in E. coli for the first time. Furthermore, we establish an efficient system for the production of biofuel 1-propanol biologically.

Rational engineering of diol dehydratase enables 1,4-butanediol biosynthesis from xylose.

Establishing novel synthetic routes for microbial production of chemicals often requires overcoming pathway bottlenecks by tailoring enzymes to enhance bio-catalysis or even achieve non-native catalysis. Diol dehydratases have been extensively studied for their interactions with C2 and C3 diols. However, attempts on utilizing these insights to enable catalysis on non-native substrates with more than two hydroxyl groups have been plagued with low efficiencies. Here, we rationally engineered the Klebsiella oxytoca diol dehydratase to enable and enhance catalytic activity toward a non-native C4 triol, 1,2,4-butanetriol. We analyzed dehydratases interaction with 1,2-propanediol and glycerol, which led us to develop rationally conceived hypotheses. An in silico approach was then developed to identify and screen candidate mutants with desired activity. This led to an engineered diol dehydratase with nearly 5 fold higher catalytic activity toward 1,2,4-butanetriol than the wild type as determined by in vitro assays. Based on this result, we then expanded the 1,2,4-butanetriol pathway to establish a novel 1,4-butanediol production platform. We engineered Escherichia colis xylose catabolism to enhance the biosynthesis of 1,2,4-butanetriol from 224mg/L to 1506mg/L. By introducing the complete pathway in the engineered strain we achieve de novo biosynthesis of 1,4-butanediol at 209mg/L from xylose. This work expands the repertoire of substrates catalyzed by diol dehydratases and serves as an elucidation to establish novel biosynthetic pathways involving dehydratase based biocatalysis.

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.

Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.

5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1-1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.

Metabolic engineering of Escherichia coli for microbial synthesis of monolignols

Monolignols are important plant metabolites involved in lignin biosynthesis. Their derivatives exhibit various physiological and pharmaceutical functions. Here, efficient enzymes were selected to construct p-coumaryl alcohol biosynthetic pathway and the titer reached 501.8±41.4mg/L under optimized conditions. The pathway was further extended to produce caffeyl alcohol and coniferyl alcohol by introducing a hydroxylase and methyltransferases. However, the promiscuity of the hydroxylase HpaBC led to the formation of an instable intermediate L-dopa from tyrosine, causing loss of the carbon sources. To solve this problem, microbial co-cultures were designed to minimize the accessibility of HpaBC to tyrosine. With the optimal inoculation ratio, 401.0±15.3mg/L of caffeyl alcohol was produced, which is nearly 12 times higher than that of the mono-culture. The titer reached 854.1±44.6mg/L in scale-up production. The same strategy was used for coniferyl alcohol production. Limited by the activity of methyltransferases, the highest titer was 124.9±5.1mg/L with 232.9±15.1mg/L of caffeyl alcohol accumulated. To the best of our knowledge, this is the first report about microbial production of caffeyl alcohol and coniferyl alcohol. This work also demonstrated the promising potential of microbial co-cultures for prevention of side-reactions.

Precursor-directed biosynthesis of 5-hydroxytryptophan using metabolically engineered E. coli.

A novel biosynthetic pathway was designed and verified reversely leading to the production of 5-hydroxytryptophan (5-HTP) from glucose. This pathway takes advantage of the relaxed substrate selectivities of relevant enzymes without employing the unstable tryptophan 5-hydroxylase. First, high-titer of 5-HTP was produced from 5-hydroxyanthranilate (5-HI) by the catalysis of E. coli TrpDCBA. Then, a novel salicylate 5-hydroxylase was used to convert the non-natural substrate anthranilate to 5-HI. After that, the production of 5-HI from glucose was achieved and optimized with modular optimization. In the end, we combined the full pathway and adopted a two-stage strategy to realize the de novo production of 5-HTP. This work demonstrated the application of enzyme promiscuity in non-natural pathway design.