Jian Hao

Isobutanol, a promising biofuel with higher energy content than ethanol, presents a sustainable alternative through biosynthesis. However, enhancing yield remains challenging due to the inefficiencies in microbial synthesis. This study introduces a transcription factor‐based biosensor using the AlkS‐PalkB system in Escherichia coli , which correlates green fluorescence with isobutanol concentration. Employing directed evolution, we modified AlkS to detect isobutanol, significantly improving biosensor specificity. Initial modifications increased the dynamic response from non‐detectable to a 2.60‐fold change. Subsequent optimisations through site‐directed mutagenesis and promoter engineering further enhanced this response to a 5.56‐fold change, equivalent to a 114% increase. Although engineered for isobutanol detection with high sensitivity, the engineered biosensor retains responsiveness to several short‐chain alcohols. This biosensor provides a foundation for high‐throughput screening of isobutanol and other short‐chain alcohol‐producing strains, though additional improvements in selectivity and operating range may be required for efficient implementation.

Background: 1,3-Propanediol (1,3-PDO) is a bulk chemical that can be produced by Klebsiella pneumoniae using glycerol as a substrate. In the 1,3-PDO synthesis pathway, part of the glycerol is oxidised to maintain intracellular NADH balance. Consequently, the theoretical maximum yield of 1,3-PDO from glycerol was lower than 1 mol/mol. Results: In this study, engineered K. pneumoniae strains were constructed to direct all glycerol toward 1,3-PDO synthesis, with NADH being supplied through the catabolism of glucose. However, glycerol utilisation was inhibited in the presence of glucose. To alleviate this carbon catabolite repression (CCR), ptsG and crr were individually knocked out. The dha pathway is responsible for 1,3-PDO synthesis. Key genes in the oxidation branch of this pathway, including dhaK , dhaL , dhaD , and gldA , were knocked out to block this pathway. However, the expression of the dha operon was impaired in these strains, resulting in low 1,3-PDO production. In contrast, knocking out dhaM , which encodes a subunit of dihydroxyacetone kinase II, effectively blocked the glycerol oxidation pathway while maintaining the activity of the dha operon. Additionally, glpK was knocked out to block the sn -glycerol-3-phosphate formation from glycerol. Glucose to glycerol with the ratio of 0.5:1 mol/mol was the optimal value for 1,3-PDO production by K. pneumoniae ∆ dhaM ∆ ptsG ∆ glpK , leading to a balance of NADH generation and consumption. Microaerobic conditions were favourable for 1,3-PDO production than anaerobic or aerobic conditions. In fed-batch fermentations, this strain produced 58.6 g/L of 1,3-PDO after 70 h, achieving a yield of 0.93 mol/mol glycerol, 2 mol/mol glucose, 0.63 mol/mol substrate. Conclusions: A highly efficient 1,3-PDO production technology that using glycerol and glucose as co-substrates was established.

绿色甲醇是生物工程领域一种新型的原料，人工甲醇营养微生物的研究成为当今的一个热点。克雷伯氏菌（Klebsiella spp.）是一类优良的工业微生物。本研究发现克雷伯氏肺炎杆菌（K. pneumoniae）不代谢甲醇，但是具有耐受高达48 g/L甲醇的能力，是一种优良的人工甲基营养底盘菌株。K. pneumoniae可以代谢甲醛，静息细胞代谢甲醛的速率达到0.15 g/L·h。为了在菌株中构建甲醇代谢途径，首先对催化甲醇反应形成甲醛的酶进行了筛选。通过对候选酶进行酶活测定和热力学分析，发现来源于黄孢原毛平革菌的O2依赖型醇氧化酶AOX具有良好催化活性。工程菌株K. pneumoniae-aox不能以甲醇为唯一碳源进行生长，但是该菌在LB培养基中36 h消耗0.94 g/L的甲醇。进一步在菌株中表达了来源于枯草芽孢杆菌的hps和phi。这两个基因编码核酮糖单磷酸代谢途径的关键酶。K. pneumoniae-aox-hps-phi在LB培养基中培养36 h消耗1.31 g/L甲醇。在以葡萄糖和甲醇为双碳源的培养基中培养K. pneumoniae-aox和K. pneumoniae-aox-hps-phi，菌株58 h分别消耗了1.70和1.39 g/L的甲醇。本研究成功构建了可以代谢甲醇的K. pneumoniae，为绿色甲醇作为原料合成化学品提供了基础。

长期以来人们认为细菌的细胞没有细胞器，但是最近二十年细菌微室的研究颠覆了这一传统观点。微室是细菌的细胞器，由壳蛋白和酶蛋白通过自组织形成。细菌微室与病毒在外观上很像，为100-200纳米的多面体。细菌微室的系统性研究源于沙门氏菌1,2-丙二醇分解代谢途径的研究。该途径相关基因形成pdu操纵子，pdu操纵子中除了酶蛋白外还有一些结构基因编码微室壳蛋白。细菌微室的生理功能是由蛋白质壳形成一个防止分子扩散的屏障，将内部环境与细胞质进行物理隔离，使得含有易挥发或有毒化合物的代谢反应容易进行。细菌微室在细菌中广泛分布，种类繁多，目前已经在分布于45个门的细菌中发现68种微室基因簇。细菌微室的生理功能具有高度多样性，在二氧化碳固定和多种化合物分解代谢中具有重要生理功能。本文主要就细菌微室的结构、生理功能、主要类别以及细菌微室在生物工程领域的应用前景等进行介绍。