Tatiana A. Pozdniakova , João P. Cruz , Paulo César Silva , Flávio Azevedo , Pier Parpot , Maria Rosario Domingues , Magnus Carlquist , Björn Johansson
{"title":"Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae","authors":"Tatiana A. Pozdniakova , João P. Cruz , Paulo César Silva , Flávio Azevedo , Pier Parpot , Maria Rosario Domingues , Magnus Carlquist , Björn Johansson","doi":"10.1016/j.mec.2023.e00224","DOIUrl":null,"url":null,"abstract":"<div><p>Fatty acids are produced by eukaryotes like baker's yeast <em>Saccharomyces cerevisiae</em> mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism <em>S. cerevisiae</em> could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (<em>FAS1</em> or <em>FAS2</em>) with a FASII consisting of nine genes from <em>Escherichia coli</em> (<em>acpP</em>, <em>acpS</em> and <em>fab</em> -<em>A</em>, -<em>B</em>, -<em>D</em>, -<em>F</em>, -<em>G</em>, -<em>H</em>, -<em>Z</em>) as well as three from <em>Arabidopsis thaliana</em> (<em>MOD1</em>, <em>FATA1</em> and <em>FATB</em>). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for <em>in-vivo</em> assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h<sup>−1</sup> without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the <em>MOD1</em> or <em>fabH</em> genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2023-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S221403012300007X","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.
期刊介绍:
Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.