Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic Engineering Communications Pub Date : 2023-06-15 DOI:10.1016/j.mec.2023.e00224
Tatiana A. Pozdniakova , João P. Cruz , Paulo César Silva , Flávio Azevedo , Pier Parpot , Maria Rosario Domingues , Magnus Carlquist , Björn Johansson
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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.

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酿酒酵母中杂交细菌/拟南芥脂肪酸合成酶系统Ⅱ的优化
脂肪酸由真核生物产生,如面包酵母酿酒酵母,主要使用大型多功能I型脂肪酸合成酶(FASI),其中七个催化步骤和一个载体结构域在一个或两个蛋白质亚基之间共享。虽然该系统可以提供催化效率,但只产生一小部分脂肪酸。原核生物、叶绿体和线粒体依赖于FAS II型(FASII),其中每个催化步骤都由一个单独基因编码的单功能酶进行。FASII更灵活,能够产生更广泛的脂肪酸结构,例如直接生产不饱和脂肪酸。在优选的工业生物酿酒酵母中高效的FASII可以为开发专门脂肪酸的可持续生产提供平台。我们用FASII在功能上取代了酵母FASI基因(FAS1或FAS2),FASII由来自大肠杆菌的9个基因(acpP、acpS和fab-a、-B、-D、-F、-G、-H、-Z)以及来自拟南芥的3个基因(MOD1、FATA1和FATB)组成。这些基因是从使用酵母通路试剂盒组装的自主复制多拷贝载体中表达的,用于在酵母中的体内组装。两轮适应使菌株在没有外源性脂肪酸的情况下具有0.19 h−1的最大生长速率(μmax),是先前报道的类似菌株生长速率的两倍。与对照相比,MOD1或fabH基因的额外拷贝导致培养物具有更高的最终细胞密度和三倍高的脂质含量。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
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.
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