PT - JOURNAL ARTICLE AU - Eline D. Postma AU - Else-Jasmijn Hassing AU - Venda Mangkusaputra AU - Jordi Geelhoed AU - Pilar de la Torre AU - Marcel van den Broek AU - Christiaan Mooiman AU - Martin Pabst AU - Jean-Marc Daran AU - Pascale Daran-Lapujade TI - Modular, synthetic chromosomes as new tools for large scale engineering of metabolism AID - 10.1101/2021.10.04.462994 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.10.04.462994 4099 - http://biorxiv.org/content/early/2021/10/05/2021.10.04.462994.short 4100 - http://biorxiv.org/content/early/2021/10/05/2021.10.04.462994.full AB - The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.Competing Interest StatementThe authors have declared no competing interest.2PE2-phenylethanolCINcinnamic acidCOCOAcoumaroyl-CoACOUMcoumaric acidDHKdihydrokaempferolK3Gkaempferol 3-O-glucosideKAEkaempferolLPEleucopelargonidinNARnaringeninNARCCnaringenin chalconeP3Gpelargonidin 3-O-glucosidePAAphenylacetic acidPELpelargonidinPHEL-phenylalaninePHLORphloretic acidpOHPPYp-hydroxyphenylpyruvatepOH2PEp-hydroxyphenylethanolpOHPAAp-hydroxyphenylacetic acidPPYphenylpyruvateTYRtyrosine3GTanthocyanin 3-O-glucosyltransferaseANSanthocyanidin synthaseC4Hcinnamate 4-hydroxylase4CL4-coumarate CoA ligaseCHIchalcone isomeraseCHSchalcone synthaseCPRcytochrome P450 reductaseDFRdihydroflavonol 4-reductaseF3Hflavanone 3-hydroxylasePALphenylalanine ammonia lyaseTALtyrosine ammonia lyaseFACSfluorescence-activated cell sortingPFEpulsed field electrophoresis