Reconstruction of the Native Biosynthetic System of Carotenoids in ─Biosynthesis of a Series of Carotenoids Specific to Paprika Fruit.

Capsanthin, capsorubin, cucurbitaxanthin A, and capsanthin 3,6-epoxide, a series of carotenoids specific to the red fruit of paprika (), were produced in pathway-engineered cells. These cells functionally expressed multiple genes for eight carotenogenic enzymes, two of which, paprika capsanthin/capsorubin synthase (CaCCS) and zeaxanthin epoxidase (CaZEP), were designed to be located adjacently. The biosynthesis of these carotenoids, except for capsanthin, was the first successful attempt in .

Use of directed enzyme evolution to create novel biosynthetic pathways for production of rare or non-natural carotenoids.

In recent years, advances in bioengineering and synthetic biology techniques have been used to create carotenoid diversity in the laboratory. In this chapter, we describe the step-by-step method to perform directed evolution of carotenoid biosynthetic enzymes. We first explain how to establish an efficient Escherichia coli colony-based screening, including a detailed description of plasmid DNA construction design as well as tips and tricks to handle and manipulate cells to produce stable colonies.

Promiscuous activity of β-carotene hydroxylase CrtZ on epoxycarotenoids leads to the formation of rare carotenoids with 6-hydroxy-3-keto-ε-ends.

Carotenoids with rare 6-hydroxy-3-keto-ε-end groups, such as piprixanthin, vitixanthin, or cochloxanthin, found in manakin birds or plants, are rare carotenoids with high antioxidant activity. The same chemical structure is found in abscisic acid or blumenol, apocarotenoids found in plants or fungi. In this study, we serendipitously discovered that the promiscuous activity of the β-carotene hydroxylase CrtZ, a diiron-containing membrane protein, can catalyze the formation of 6-hydroxy-3-keto-ε-end by using epoxycarotenoids antheraxanthin or violaxanthin as substrate.

Capsanthin Production in by Overexpression of Capsanthin/Capsorubin Synthase from .

Capsanthin, a characteristic red carotenoid found in the fruits of red pepper (), is widely consumed as a food and a functional coloring additive. An enzyme catalyzing capsanthin synthesis was identified as capsanthin/capsorubin synthase (CCS) in the 1990s, but no microbial production of capsanthin has been reported. We report here the first successful attempt to biosynthesize capsanthin in by carotenoid-pathway engineering.

Genetic Encoding of Targeted Magnetic Resonance Imaging Contrast Agents for Tumor Imaging.

Tumor-selective contrast agents have the potential to aid in the diagnosis and treatment of cancer using noninvasive imaging modalities such as magnetic resonance imaging (MRI). Such contrast agents can consist of magnetic nanoparticles incorporating functionalities that respond to cues specific to tumor environments. Genetically engineering magnetotactic bacteria to display peptides has been investigated as a means to produce contrast agents that combine the robust image contrast effects of magnetosomes with the transgenic-targeting peptides displayed on their surface.

Nonnatural biosynthetic pathway for 2-hydroxylated xanthophylls with C-carotenoid backbone.

Carotenoids are structurally diverse pigments with various important biological functions. There has been a large interest in the search for novel carotenoid structures, since only a slight structural changes can result in a drastic difference in their biological functions. Carotenoid-modifying enzymes show remarkable substrate promiscuity, allowing rapid access to a vast set of novel carotenoids by combinatorial biosynthesis.

Genetically engineered biosynthetic pathways for nonnatural C carotenoids using C-elongases and C-cyclases in Escherichia coli.

While the majority of the natural carotenoid pigments are based on 40-carbon (C) skeleton, some carotenoids from bacteria have larger C skeleton, biosynthesized by attaching two isoprene units (C) to both sides of the C carotenoid pigment lycopene. Subsequent cyclization reactions result in the production of C carotenoids with diverse and unique skeletal structures. To produce even larger nonnatural novel carotenoids with C + C + C = C skeletons, we systematically coexpressed natural C carotenoid biosynthetic enzymes (lycopene C-elongases and C-cyclases) from various bacterial sources together with the laboratory-engineered nonnatural C-lycopene pathway in Escherichia coli.

Construction of a Nonnatural C Carotenoid Biosynthetic Pathway.

Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C carotenoids by engineering a C-carotenoid backbone synthase (CrtM) from Staphylococcus aureus.

Directed evolution of the autoinducer selectivity of Vibrio fischeri LuxR.

LuxR family transcriptional regulators are the core components of quorum sensing in Gram-negative bacteria and exert their effects through binding to the signaling molecules acyl-homoserine lactones (acyl-HSLs). The function of the LuxR homologs is remarkably plastic, and naturally occurring acyl-HSLs are structurally diverse. To investigate the molecular basis of the functional plasticity of Vibrio fischeri LuxR, we directed the evolution of LuxR toward three different specificities in the laboratory.

A highly selective biosynthetic pathway to non-natural C50 carotenoids assembled from moderately selective enzymes.

Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid.

Production of squalene by squalene synthases and their truncated mutants in Escherichia coli.

Squalene is a precursor of thousands of bioactive triterpenoids and also has industrial value as a lubricant, health-promoting agent, and/or drop-in biofuel. To establish an efficient Escherichia coli-based system for squalene production, we tested two different squalene synthases and their mutants in combination with precursor pathways. By co-expressing a chimeric mevalonate pathway with human or Thermosynechococcus squalene synthase, E.

Directed evolution of squalene synthase for dehydrosqualene biosynthesis.

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C30 carotenoid backbone, dehydrosqualene.

A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis.

Construction of carotenoid biosynthetic pathways using squalene synthase.

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C30 carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C30 carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a "squalene route" and a "lycopersene route" for C30 and C40 carotenoids, respectively.

Evolutionary analysis of the functional plasticity of Staphylococcus aureus C30 carotenoid synthase.

Most natural carotenoids have 40-carbon (C40) backbones, while some bacteria produce carotenoids with C30 backbones. Carotenoid backbone synthases, the enzyme that catalyze the first committed step in carotenoid biosynthesis, are known to be highly specific. Previously, using C30 backbone synthase (diapophytoene synthase, CrtM) from Staphylococcus aureus, we reported two size-shifting mutations, F26A and W38A, which confer C40 synthase activity at the cost of the original C30 synthase activity.

Directed evolution of carotenoid synthases for the production of unnatural carotenoids.

Directed evolution is a well-established strategy to confer novel catalytic functions to the enzymes. Thanks to the relative ease of establishing color screening, carotenogenic enzymes can be rapidly evolved in the laboratory for novel functions. The combinatorial usages of the evolvants result in the creation of diverse set of novel, sometimes unnatural carotenoids.