Carbon feedstocks from fossilized sources are being rapidly depleted due to rising demand for industrial and commercial applications. Many petroleum-derived chemicals can be directly or functionally substituted with chemicals derived from renewable feedstocks. Several short chain organic acids may fulfill this role using their functional groups as a target for chemical catalysis. Saccharomyces cerevisiae was engineered to produce short chain carboxylic acids (C6 to C10 ) from glucose using the heterologous Homo sapiens type I fatty acid synthase (hFAS). This synthase was activated by phosphopantetheine transfereases AcpS and Sfp from Escherichia coli and Bacillus subtilis, respectively, both in vitro and in vivo. hFAS was produced in the holo-form and produced carboxylic acids in vitro, confirmed by NADPH and ADIFAB assays. Overexpression of hFAS in a yeast FAS2 knockout strain, deficient in de novo fatty acid synthesis, demonstrated the full functional replacement of the native fungal FAS by hFAS. Two active heterologous short chain thioesterases (TEs) from Cuphea palustris (CpFatB1) and Rattus norvegicus (TEII) were evaluated for short chain fatty acid (SCFA) synthesis in vitro and in vivo. Three hFAS mutants were constructed: a mutant deficient in the native TE domain, a mutant with a linked CpFatB1 TE and a mutant with a linked TEII TE. Using the native yeast fatty acid synthase for growth, the overexpression of the hFAS mutants and the short-chain TEs (linked or plasmid-based) increased in vivo caprylic acid and total SCFA production up to 64-fold (63 mg/L) and 52-fold (68 mg/L), respectively, over the native yeast levels. Combined over-expression of the phosphopantetheine transferase with the hFAS mutant resulted in C8 titers of up to 82 mg/L and total SCFA titers of up to 111 mg/L.

译文

由于工业和商业应用需求的增长,化石来源的碳原料正在迅速耗尽。许多石油衍生的化学物质可以直接或功能上被可再生原料衍生的化学物质替代。几种短链有机酸可以使用其官能团作为化学催化的目标来发挥这一作用。酿酒酵母经过工程改造,可使用异源智人I型脂肪酸合酶 (hFAS) 从葡萄糖中产生短链羧酸 (C6至C10)。在体外和体内,该合酶分别由来自大肠杆菌和枯草芽孢杆菌的磷酸核苷转染酶AcpS和Sfp激活。通过NADPH和ADIFAB分析证实,hFAS以全型生产并在体外生产羧酸。在缺乏从头脂肪酸合成的酵母FAS2敲除菌株中hFAS的过表达证明了hFAS对天然真菌FAS的完全功能替代。在体外和体内评估了来自Cuphea palustris (CpFatB1) 和Rattus norvegicus (TEII) 的两种活性异源短链硫酯酶 (TEs) 的短链脂肪酸 (SCFA) 合成。构建了三个hFAS突变体: 天然TE结构域缺陷的突变体,CpFatB1 TE连接的突变体和TEII TE连接的突变体。使用天然酵母脂肪酸合酶进行生长,hFAS突变体和短链TEs (连接或基于质粒) 的过表达增加了体内辛酸和总SCFA产量高达64倍 (63  mg/L) 和52倍 (68  mg/L),分别超过天然酵母水平。磷酸泛氨酸转移酶与hFAS突变体的联合过表达导致C8滴度高达82  mg/L,总SCFA滴度高达111  mg/L。

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