Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway

Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.

短链脂肪酸（Short-chain fatty acids, SCFAs）如丁酸，在化工与燃料工业中拥有广泛应用。全球对可持续燃料与化学品的旺盛需求，推动研究者致力于通过微生物途径合成SCFAs。本研究选取三种硫酯酶（thioesterases）开展对比研究：来自四联厌氧球菌（Anaerococcus tetradius）的TesAT、来自产甲酸布氏菌（Bryantella formatexigens）的TesBF，以及来自多形拟杆菌（Bacteroides thetaiotaomicron）的TesBT，在大肠杆菌（Escherichia coli）中利用内源脂肪酸合成（FASII）途径生产SCFAs，并通过调控遗传与生物工艺参数以提升产物得率与生产强度。携带tesBT基因的重组大肠杆菌菌株丁酸效价最高，达1.46 g·L⁻¹；其次为携带tesBF的菌株（0.85 g·L⁻¹），携带tesAT的菌株效价最低，为0.12 g·L⁻¹。丁酸效价随质粒拷贝数与菌株基因型的不同呈现显著差异。针对已知可影响长链脂肪酸合成的遗传因子进行调控，例如敲除启动脂肪酸降解循环的fadD与fadE基因，以及过表达作为脂肪酸生物合成全局转录激活因子且可抑制降解循环的fadR基因，均未显著提升丁酸效价。使用可抑制脂肪酸延伸循环的化学抑制剂浅蓝菌素（cerulenin），可使TesBF菌株的丁酸效价提升1.7倍，但对TesBT菌株却产生不利影响。体外酶活实验显示，浅蓝菌素亦可抑制短链特异性硫酯酶，但其抑制浓度因硫酯酶类型不同而存在差异。在此基础上进一步优化工艺，并结合限磷条件下的补料分批培养，最终实现丁酸效价14.3 g·L⁻¹、总游离脂肪酸效价17.5 g·L⁻¹，对应理论得率28%。本研究拓展了学界对大肠杆菌通过FASII途径生产SCFAs的认知，并凸显了遗传与工艺优化对目标产物提升的关键作用。