Genome-scale metabolic network models have been successfully applied to guide metabolic engineering. However, the conventional flux balance analysis only considers stoichiometry and reaction direction constraints, and the simulation results cannot accurately describe certain phenomena such as overflow metabolism and diauxie growth on two substrates. Recently, researchers proposed new constraint-based methods to simulate the cellular behavior under different conditions more precisely by introducing new constraints such as limited enzyme content and thermodynamics feasibility. Here we review several enzyme-constrained models, giving a comprehensive introduction on the biological basis and mathematical representation for the enzyme constraint, the optimization function, the impact on the calculated flux distribution and their application in identification of metabolic engineering targets. The main problems in these existing methods and the perspectives on this emerging research field are also discussed. By introducing new constraints, metabolic network models can simulate and predict cellular behavior under various environmental and genetic perturbations more accurately, and thus can provide more reliable guidance to strain engineering.
基因组尺度代谢网络模型已经成功地应用于指导代谢工程改造，但由于传统通量平衡分析法仅考虑化学计量学和反应方向约束，模拟得到的是理论最优结果，对一些现象如代谢溢流、底物层级利用等无法准确描述。近年来人们通过在代谢网络模型中引入新的蛋白量、热力学等约束发展了新的约束优化计算方法，可以更准确真实地模拟细胞在不同条件下的代谢行为。文中主要对近年来提出的多种酶约束模型进行评述，对酶约束引入的基本思路、酶约束的数学方程表示及优化目标设定、引入酶约束后对代谢通量计算结果的影响及酶约束模型在代谢工程菌种改造中的应用等进行了全面深入的介绍，并提出了已有各种方法存在的主要问题，展望了相关方法的未来发展方向。通过引入新的约束，代谢网络模型能够更精确模拟和预测细胞在环境和基因扰动下的代谢行为，为代谢工程菌种改造提供更准确可靠的指导。.
Keywords: enzyme constraints; enzyme kinetics; metabolic engineering; metabolic network model; thermodynamics.