Non-natural Pathway Research Articles

Production of Gibberellins via a Non-Natural Pathway Using Steviol as a Substrate.

Gibberellins (GAs) are plant hormones widely used in agriculture. At present, GAs are produced by fermentation of the fungus Fusarium fujikuroi. However, fungal growth is too slow, resulting in slow fungal fermentation and a low yield. Here, to develop an alternative production source of GAs, an artificial pathway was engineered in Escherichia coli. By selecting and combining enzymes derived from plants and bacteria, a novel 4-enzyme pathway was successfully constructed to produce GAs using steviol, a readily available and less valuable byproduct during enzymatic refining of rebaudioside A, as a feedstock. Whole-cell biotransformation with E. coli strain expressing the novel pathway produced 71.17 ± 2.00 mg/L GA1 and a trace amount of GA3 from steviol in 48 h. This report presents a significant advancement in the fast production of GAs and establishes a method for the metabolism of terpenoids to produce target products in microbial hosts.

De Novo Biosynthesis of D-p-Hydroxyphenylglycine by a Designed Cofactor Self-Sufficient Route and Co-culture Strategy.

d-p-Hydroxyphenylglycine (D-HPG) is an important intermediate for the synthesis of β-lactam antibiotics with an annual market demand of thousands of tons. Currently, the main production processes are via chemical approaches. Although enzymatic conversion has been investigated for D-HPG production, synthesis of the substrate DL-hydroxyphenylhydantoin is still chemically based, which suffers from high pollution and harsh reaction conditions. In this study, one cofactor self-sufficient route for D-HPG production from l-phenylalanine was newly designed and the artificial pathway was functionalized by selecting suitable enzymes and adjusting their expressions in strain Pseudomonas putida KT2440. Notably, a new R-mandelate dehydrogenase from Lactococcus lactis with relatively high activity under pH neutral conditions was successfully mined to demonstrate the biosynthetic pathway in vivo. The performance of the engineered P. putida strain was further increased by enhancing cellular NAD availability and blocking l-phenylalanine consumption. Coupled with the l-phenylalanine producer, Escherichia coli strain ATCC 31884, a stable and interactive co-culture process was also developed by engineering a "cross-link auxotrophic" system to produce D-HPG directly from glucose. Thus, this study is the first approach for the de novo biosynthesis of D-HPG by engineering a non-natural pathway and lays the foundation for further improving the efficiency of D-HPG production via a green and sustainable route.

Efficient Production of 1,3-Propanediol from Diverse Carbohydrates via a Non-natural Pathway Using 3-Hydroxypropionic Acid as an Intermediate.

1,3-Propanediol (1,3-PDO) is a promising platform chemical used to manufacture various polyesters, polyethers, and polyurethanes. Microbial production of 1,3-PDO using non-natural producers often requires adding expensive cofactors such as vitamin B12, which increases the whole production cost. In this study, we proposed and engineered a non-natural 1,3-PDO synthetic pathway derived from acetyl-CoA, enabling efficient accumulation of 1,3-PDO in Escherichia coli without adding expensive cofactors. This functional pathway was established by introducing the malonyl-CoA-dependent 3-hydroxypropionic acid (3-HP) module and screening the key enzymes to convert 3-HP to 1,3-PDO. The best engineered strain can produce 2.93 g/L 1,3-PDO with a yield of 0.35 mol/mol glucose in shake flask cultivation (and 7.98 g/L in fed-batch fermentation), which is significantly higher than previous reports based on homoserine- or malate-derived non-natural pathways. We also demonstrated for the first time the feasibility of producing 1,3-PDO from diverse carbohydrates including xylose, glycerol, and acetate based on the same pathway. Thus, this study provides an alternative route for 1,3-PDO production.

Metabolic Engineering of Escherichia coli for De Novo Production of 1,5-Pentanediol from Glucose.

1,5-Pentanediol (1,5-PDO) is an important C5 building block for the synthesis of different value-added polyurethanes and polyesters. However, no natural metabolic pathway exists for the biosynthesis of 1,5-PDO. Herein we designed and constructed a promising nonnatural pathway for de novo production of 1,5-PDO from cheap carbohydrates. This biosynthesis route expands natural lysine pathways and employs two artificial metabolic modules to sequentially convert lysine into 5-hydroxyvalerate (5-HV) and 1,5-PDO via 5-hydroxyvaleryl-CoA. Theoretically, the 5-hydroxyvaleryl-CoA-based pathway is more energy-efficient than a recently published carboxylic acid reductase-based pathway for 1,5-PDO production. By combining strategies of systematic enzyme screening, pathway balancing, and transporter engineering, we successfully constructed a minimally engineered Escherichia coli strain capable of producing 3.19 g/L of 5-HV and 0.35 g/L of 1,5-PDO in a medium containing 20 g/L of glucose and 5 g/L lysine. Introducing the synthetic modules into a lysine producer and enhancing NADPH supply enabled the strain to accumulate 1.04 g/L of 5-HV and 0.12 g/L of 1,5-PDO using glucose as the main carbon source. This work lays the basis for the development of a biological route for 1,5-PDO production from renewable bioresources.

A New Synthetic Pathway for the Bioproduction of Glycolic Acid From Lignocellulosic Sugars Aimed at Maximal Carbon Conservation.

Glycolic acid is a two-carbon α-hydroxy acid with many applications in industrial sectors including packaging, fine chemistry, cosmetics, and pharmaceutics. Currently, glycolic acid is chemically manufactured from fossil resources. This chemical mode of production is raising some concerns regarding its use in health for personal care. Microbial production of GA stands as a remarkable challenge to meet these concerns, while responding to the increasing demand to produce bio-sourced products from renewable carbon resources. We here report on the design and expression of a novel non-natural pathway of glycolic acid in E. coli. The originality of this new pathway, termed “glycoptimus” relies on two pillars. On the one hand, it requires the overexpression of three naturally occurring E. coli genes, namely kdsD encoding a D-arabinose-5-P isomerase, fsaA encoding a class 1 aldolase that cleaves D-arabinose-5-P into glyceraldehyde-3-P and glycolaldehyde, and aldA coding for an aldehyde dehydrogenase that oxidizes glycoladehyde in glycolate. These three genes constitute the “glycoptimus module.” On the other hand, the expression of these genes together with a reshaping of the central carbon metabolism should enable a production of glycolic acid from pentose and hexose at a molar ratio of 2.5 and 3, respectively, which corresponds to 50% increase as compared to the existing pathways. We demonstrated the ‘in vivo’ potentiality of this pathway using an E. coli strain, which constitutively expressed the glycoptimus module and whose carbon flow in glycolysis was blocked at the level of glyceraldehyde-3-P dehydrogenase reaction step. This engineered strain was cultivated on a permissive medium containing malate and D-glucose. Upon exhaustion of malate, addition of either D-glucose, D-xylose or L-arabinose led to the production of glycolic acid reaching about 30% of the maximum molar yield. Further improvements at the level of enzymes, strains and bioprocess engineering are awaited to increase yield and titer, rendering the microbial production of glycolic acid affordable for a cost-effective industrial process.

Construction of a synthetic pathway for the production of 1,3-propanediol from glucose

In this work, we describe the construction of a synthetic metabolic pathway enabling direct biosynthesis of 1,3-propanediol (PDO) from glucose via the Krebs cycle intermediate malate. This non-natural pathway extends a previously published synthetic pathway for the synthesis of (L)-2,4-dihydroxybutyrate (L-DHB) from malate by three additional reaction steps catalyzed respectively, by a DHB dehydrogenase, a 2-keto-4-hydroxybutyrate (OHB) dehydrogenase and a PDO oxidoreductase. Screening and structure-guided protein engineering provided a (L)-DHB dehydrogenase from the membrane-associated (L)-lactate dehydrogenase of E. coli and OHB decarboxylase variants derived from the branched-chain keto-acid decarboxylase encoded by kdcA from Lactococcus lactis or pyruvate decarboxylase from Zymomonas mobilis. The simultaneous overexpression of the genes encoding these enzymes together with the endogenous ydhD-encoded aldehyde reductase enabled PDO biosynthesis from (L)-DHB. While the simultaneous expression of the six enzymatic activities in a single engineered E. coli strain resulted in a low production of 0.1 mM PDO from 110 mM glucose, a 40-fold increased PDO titer was obtained by co-cultivation of an E. coli strain expressing the malate-DHB pathway with another strain harboring the DHB-to-PDO pathway.

Metabolic Engineering of a Homoserine-Derived Non-Natural Pathway for the De Novo Production of 1,3-Propanediol from Glucose.

Engineering a homoserine-derived non-natural pathway allows heterologous production of 1,3-propanediol (1,3-PDO) from glucose without adding expensive vitamin B12. Due to the lack of efficient enzymes to catalyze the deamination of homoserine and the decarboxylation of 4-hydroxy-2-ketobutyrate, the previously engineered strain can only produce 51.5 mg/L 1,3-PDO using homoserine and glucose as cosubstrates. In this study, we systematically screened the enzymes from different protein families to catalyze the two corresponding reactions and further optimized the selected enzymes by protein engineering. Together with the improvement of homoserine supply by systematic metabolic engineering, an engineered Escherichia coli strain with an optimal combination of aspartate transaminase ( aspC) from E.coli, pyruvate decarboxylase ( pdc) from Zymomonas mobiliz, and alcohol dehydrogenase yqhD from E.coli can produce 0.32 g/L 1,3-PDO from glucose in shake flask cultivation. The titer of 1,3-PDO was further increased to 0.49 g/L or 0.63 g/L by introducing a point mutation of I472A into pdc gene or constructing a fusion protein between aspC and pdc. This study lays the basis for developing a potential process for 1,3-PDO production from sugars without using expensive coenzyme B12.

Engineering Escherichia coli for renewable benzyl alcohol production

Benzyl alcohol is an aromatic hydrocarbon used as a solvent and an intermediate chemical in the pharmaceutical, cosmetics, and flavor/fragrance industries. The de novo biosynthesis of benzyl alcohol directly from renewable glucose was herein explored using a non-natural pathway engineered in Escherichia coli. Benzaldehyde was first produced from endogenous phenylpyruvate via three heterologous steps, including hydroxymandelate synthase (encoded by hmaS) from Amycolatopsis orientalis, followed by (S)-mandelate dehydrogenase (encoded by mdlB) and phenylglyoxylate decarboxylase (encoded by mdlC) from Pseudomonas putida ATCC 12633. The subsequent rapid and efficient reduction of benzaldehyde to benzyl alcohol occurred by the combined activity and native regulation of multiple endogenous alcohol dehydrogenases and/or aldo-keto reductases. Through systematic deletion of competing aromatic amino acid biosynthesis pathways to promote endogenous phenylpyruvate availability, final benzyl alcohol titers as high as 114±1mg/L were realized, representing a yield of 7.6±0.1mg/g on glucose and a ~5-fold improvement over initial strains.

Fully glycerol-independent microbial production of 1, 3-propanediol via non-natural pathway: paving the way to success with synthetic tiles.

Fully glycerol-independent microbial production of 1, 3-propanediol via non-natural pathway: paving the way to success with synthetic tiles.

Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose.

Protein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non-natural pathways. No natural microorganism can directly use sugars to produce 1,3-propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4-hydroxy-2-ketobutyrate and 3-hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non-natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4-hydroxy-2-ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products.

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.

ATP drives direct photosynthetic production of 1-butanol in cyanobacteria

While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature and has been demonstrated in various organisms, the first step of the pathway, condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically unfavorable. Here, we show that artificially engineered ATP consumption through a pathway modification can drive this reaction forward and enables for the first time the direct photosynthetic production of 1-butanol from cyanobacteria Synechococcus elongatus PCC 7942. We further demonstrated that substitution of bifunctional aldehyde/alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased 1-butanol production by 4-fold. These results demonstrated the importance of ATP and cofactor driving forces as a design principle to alter metabolic flux.

Etioplast and etio-chloroplast formation under natural conditions: the dark side of chlorophyll biosynthesis in angiosperms

Chloroplast development is usually regarded as proceeding from proplastids. However, direct or indirect conversion pathways have been described in the literature, the latter involving the etioplast or the etio-chloroplast stages. Etioplasts are characterized by the absence of chlorophylls (Chl-s) and the presence of a unique inner membrane network, the prolamellar body (PLB), whereas etio-chloroplasts contain Chl-s and small PLBs interconnected with chloroplast thylakoids. As etioplast development requires growth in darkness for several days, this stage is generally regarded as a nonnatural pathway of chloroplast development occurring only under laboratory conditions. In this article, we have reviewed the data in favor of the involvement of etioplasts and etio-chloroplasts as intermediary stage(s) in chloroplast formation under natural conditions, the molecular aspects of PLB formation and we propose a dynamic model for its regulation.

Biosynthesis of novel thermoplastic polythioesters by engineered Escherichia coli.

The development of non-petrochemical sources for the plastics industry continues to progress as large multinationals focus on renewable resources to replace fossil carbon. Many bacteria are known to accumulate polyoxoesters as water-insoluble granules in the cytoplasm. The thermoplastic and/or elastomeric behaviour of these biodegradable polymers holds promise for the development of various technological applications. Here, we report the synthesis and characterization of microbial polythioesters (PTEs), a novel class of biopolymers of general technological relevance. Biosynthesis of PTE homopolymers was achieved using a recombinant strain of Escherichia coli that expressed a non-natural pathway consisting of a butyrate kinase, a phosphotransbutyrylase, and a PHA synthase. Different homopolymers were produced, consisting of either 3-mercaptopropionate, 3-mercaptobutyrate, or 3-mercaptovalerate repeating units, if the respective mercaptoalkanoic acids were provided as precursor substrates to the fermentative process. The PTEs contributed up to 30% (w/w) of the cellular dry weight and were identified as hydrophobic inclusions in the cytoplasm. The chemical and stereochemical homogeneity of the purified PTEs were identified by different methods, and the estimated physical properties were compared to the oxypolyester equivalents, revealing low crystalline order and, for the poly(3-mercaptopropionate) improved thermal stability. The ability to produce PTEs through a biosynthetic route opens up new avenues in the field of biomaterials.