Pyung Cheon Lee

Metabolic engineering towards biotechnological production of carotenoids in microorganisms

Abstract. Carotenoids are important natural pigments produced by many microorganisms and plants. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. The recent discoveries of health-related beneficial properties attributed to carotenoids have spurred great interest in the production of structurally diverse carotenoids for pharmaceutical applications. The availability of a considerable number of microbial and plant carotenoid genes that can be functionally expressed in heterologous hosts has opened ways for the production of diverse carotenoid compounds in heterologous systems. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity. In addition, we will discuss the application of combinatorial and evolutionary strategies to carotenoid pathway engineering to broaden the diversity of carotenoid structures synthesized in recombinant hosts.

Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli

BackgroundPhenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells.ResultsA pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure.ConclusionPhenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

Exploring Recombinant Flavonoid Biosynthesis in Metabolically Engineered Escherichia coli

Flavonoids are important plant‐specific secondary metabolites synthesized from 4‐coumaroyl coenzyme A (CoA), derived from the general phenylpropanoid pathway, and three malonyl‐CoAs. The synthesis involves a plant type III polyketide synthase, chalcone synthase. We report the cloning and coexpression in Escherichia coli of phenylalanine ammonia lyase, cinnamate‐4‐hydroxylase, 4‐coumarate:CoA ligase, and chalcone synthase from the model plant Arabidopsis thaliana. Simultaneous expression of all four genes resulted in a blockage after the first enzymatic step caused by the presence of nonfunctional cinnamate‐4‐hydroxylase. To overcome this problem we fed exogenous 4‐coumaric acid to induced cultures. We observed high‐level production of the flavanone naringenin as a result. We were also able to produce phloretin by feeding cultures with 3‐(4‐hydroxyphenyl)propionic acid. Feeding with ferulic or caffeic acid did not yield the corresponding flavanones. We have also cloned and partially characterized a new tyrosine ammonia lyase from Rhodobacter sphaeroides. Tyrosine ammonia lyase was substituted for phenylalanine ammonia lyase and cinnamate‐4‐hydroxylase in our E. coli clones and three different growth media were tested. After 48 h induction, high‐level production (20.8 mgu2009L−1) of naringenin in metabolically engineered E. coli was observed for the first time.

Biosynthesis of structurally novel carotenoids in Escherichia coli

Previously, we utilized in vitro evolution to alter the catalytic functions of several carotenoid enzymes and produce the novel carotenoids tetradehydrolycopene and torulene in Escherichia coli. Here we report on the successful extension of these pathways and the C(30) carotenoid diaponeurosporene pathway with additional carotenoid genes. Extension of the known acyclic C(30) pathway with C(40) carotenoid enzymes-spheroidene monooxygenase and lycopene cyclase-yielded new oxygenated acylic products and the unnatural cyclic C(30) diapotorulene, respectively. Extension of acyclic C(40) pathways with spheroidene monooxygenase generated novel oxygenated carotenoids including the violet phillipsiaxanthin. Extension of the torulene biosynthetic pathway with carotene hydroxylase, desaturase, glucosylase, and ketolase yielded new torulene derivatives. These results demonstrate the utility of extending an in vitro evolved central metabolic pathway with catalytically promiscuous downstream enzymes in order to generate structurally novel compounds.

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli.

Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28°C showed better production of torulene than 37°C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2×YT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and β-carotene, in E. coli. In contrast, glycerol-containing LB, 2×YT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1–3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.

Biosynthesis of ubiquinone compounds with conjugated prenyl side chains

ABSTRACT Enzymatic steps from two different biosynthetic pathways were combined in Escherichia coli, directing the synthesis of a new class of biomolecules—ubiquinones with prenyl side chains containing conjugated double bonds. This was achieved by the activity of a C30 carotenoid desaturase, CrtN, from Staphylococcus aureus, which exhibited an inherent flexibility in substrate recognition compared to other carotenoid desaturases. By utilizing the known plasticity of E. colis native ubiquinone biosynthesis pathway and the unusual activity of CrtN, modified ubiquinone structures with prenyl side chains containing conjugated double bonds were generated. The side chains of the new structures were confirmed to have different degrees of desaturation by mass spectrometry and nuclear magnetic resonance analysis. In vivo 14C labeling and in vitro activity studies showed that CrtN desaturates octaprenyl diphosphates but not the ubiquinone compounds directly. Antioxidant properties of conjugated side chain ubiquinones were analyzed in an in vitro β-carotene-linoleate model system and were found to be higher than the corresponding unmodified ubiquinones. These results demonstrate that by combining pathway steps from different branches of biosynthetic networks, classes of compounds not observed in nature can be synthesized and structural motifs that are functionally important can be combined or enhanced.

Creating Carotenoid Diversity in E. coli Cells using Combinatorial and Directed Evolution Strategies

Carotenoids represent a structurally diverse class of pigments with important biological functions and commercial applications. Biosynthesis of carotenoids has been studied on a molecular level for the core pathways and recombinant hosts have been engineered for heterologous carotenoid production. This paper summarizes our efforts on accessing novel carotenoid compounds in engineered E. coli by altering the catalytic activities of enzymes using in vitro evolution, by exploring the catalytic promiscuity of known carotenoid enzymes and by mining for novel enzymes in microbial genome sequences.