Butanol Challenge of L. brevis . Levilactobacillus brevis

The presence of anti-microbial phenolic compounds, such as the model compound ferulic acid, in biomass hydrolysates poses significant challenges to the widespread use of biomass in conjunction with whole cell biocatalysis or fermentation. Biofuel toxicity must also be overcome to allow for efficient production of next generation biofuels such as butanol, isopropanol, and others for widespread usage. Currently, these inhibitory compounds must be removed through additional downstream processing or sufficiently diluted to create environments suitable for most industrially important microbial strains. This study explores the high ferulic acid and n-butanol tolerance in Lactobacillus brevis (L. brevis), a lactic acid bacteria often found in fermentation processes, by global transcriptional response analysis. The transcriptional profile of L. brevis under ferulic acid and butanol stress reveals that the presence of ferulic acid primarily triggers the expression of membrane proteins to counteract ferulic acid induced changes in membrane fluidity and ion leakage. In contrast to the ferulic acid stress response, butanol addition to growing cultures uniquely induced the entire fatty acid synthesis pathway in the midst of a generalized stress response. Overexpression of the rate-limiting acetyl-CoA carboxylase subunits (AccABCD) in E. coli to increase lipid synthesis had no effect on butanol tolerance, suggesting that additional engineering is necessary to produce sufficient levels of appropriate fatty acids to confer butanol tolerance. Several promising routes for understanding both phenolic acid and butanol tolerance have been identified based upon these findings. These insights may be used to guide further engineering of model industrial organisms to better tolerate both classes of inhibitors in processed biomass used for biofuel production. Overall design: Cultures were grown to OD ~ 0.2 in MRS media (baffled flasks), T = 30 C, 100 rpm. Butanol was then added to the cultures. Samples were harvested 15, 75, and 135 min after butanol addition. Each time point has 3 biological replicates, and dye swaps were incorporated into the microarray experiments.

生物质水解液中存在抗菌酚类化合物，如模型化合物阿魏酸（ferulic acid），会严重制约生物质与全细胞生物催化（whole cell biocatalysis）、发酵技术的规模化应用。此外，还需克服生物燃料毒性壁垒，以实现丁醇、异丙醇等新一代生物燃料的高效量产并推动其广泛应用。当前，这类抑制性化合物需通过额外的下游工艺去除，或是经充分稀释后，才能为多数工业常用微生物菌株营造适宜的生长环境。本研究通过全局转录组响应分析，探究了发酵过程中常见的乳酸菌（lactic acid bacteria）——短乳杆菌（Lactobacillus brevis, L. brevis）对高浓度阿魏酸与正丁醇的耐受性。对阿魏酸和丁醇胁迫下短乳杆菌的转录组分析显示，阿魏酸主要诱导膜蛋白的表达，以抵消其引发的膜流动性改变与离子渗漏问题。与阿魏酸胁迫响应不同，向生长中的培养物添加丁醇后，会在广谱胁迫响应过程中特异性激活整条脂肪酸合成通路。在大肠杆菌（E. coli）中过表达限速型乙酰辅酶A羧化酶亚基（AccABCD）以提升脂质合成的策略，并未对丁醇耐受性产生显著提升效果，这表明需开展额外的工程化改造，以合成足量适配性脂肪酸，从而赋予菌株丁醇耐受性。基于上述研究结果，已明确多条阐释酚酸与丁醇耐受性的可行研究路径。这些研究成果可用于指导模式工业菌株的进一步工程化改造，使其能够更好地耐受生物燃料生产所用预处理生物质中的两类抑制物。整体实验设计：将菌株在MRS培养基（MRS media）中培养至光密度（optical density, OD）约0.2，使用带挡板摇瓶于30℃、100 rpm条件下进行培养。随后向培养物中添加丁醇，分别于添加丁醇后的15 min、75 min、135 min收集样本。每个时间点设置3次生物学重复，且基因芯片（microarray）实验中纳入了染料互换设计。