Redesign, Reconstruction, and Directed Extension of the Brevibacterium linens C ₄₀ Carotenoid Pathway in Escherichia coli

Abstract

In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY 2 and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed ␤-carotene pathway synthesized an unexpected 7,8-dihydro-␤-carotene in addition to ␤-carotene. Extension of the ␤-carotene pathway with the B. linens ␤-ring desaturase CrtU and Pantoea ␤-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.

Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

“…C1B1Y (AY876938) (see Table S1 in the supplemental material). The PCR products were then cloned into pUCM (15), which has been modified to facilitate the constitutive expression of a cloned gene, resulting in pUCM-X PAG (where X is a pathway gene) ( Table 1).…”

“…To assemble the carotenogenic pathway genes of P. agglomerans, genes together with a modified constitutive lac-promoter and a ribosome binding site were sequentially subcloned from pUCM-X (where X is a pathway gene) into pACYC184 vector as described previously (15). Briefly, a CrtE expression module was subcloned from pUCM-E PAG into the HindIII site of pACYC184, resulting in pACM-E PAG ; a CrtB expression module was subcloned from pUCM-B PAG into the SalI site of pACYC184, resulting in pACM-B PAG ; and a CrtI expression module was subcloned from pUCM-I PAG into the BamHI site of pACYC184, resulting in pACM-I PAG (Table 1).…”

“…The carotenoid biosynthetic pathways of Pantoea have been successfully used as basic pathways for many carotenoid studies (11)(12)(13). The pathway enzymes from different sources have occasionally shown unusual activities or substrate affinities in a heterologous host (14)(15)(16). Carotenogenic enzymes, especially carotenoid-modifying enzymes, tend to be promiscuous in their substrate specificity (14) and show unexpected/hidden activities (3,16) when expressed in heterologous host microorganisms.…”

Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus : Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C 50 Carotenoid Cyclases

“…In particular, our studies have generated new and important insight into the diverse catalytic functions of natural carotenoid cyclases as a novel class of enzymes directing biosynthesis of structurally different C 50 carotenoids. Recently, the C 40 carotenoid biosynthetic pathway of Brevibacterium linens was redesigned and extended by recruitment of heterologous genes leading to production of unexpected carotenoids in E. coli hosts (19). As we see it, C 50 cyclases should represent interesting targets for future synthetic biology approaches aiming at generating structurally diverse carotenoids with interesting properties that might not be present in nature.…”

Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli

“…Genes encoding CrtM, CrtN, CrtP, CrtQ, and CrtO from S. aureus ssp. aureus (KCTC 1928) were cloned into the constitutive expression vector pUCM (22). Four genes, encoding AldH (aldH), glycine betaine aldehyde dehydrogenase (gbsA), aldehyde dehydrogenase homolog (aldA), and aldehyde dehydrogenase family protein, were amplified from the genomic DNA of S. aureus KCTC 1928 with PCR primers that were designed according to corresponding gene sequences from S. aureus strain Newman (AP009351) and cloned into the pUCM vector.…”