谷氨酸废液对枯草芽孢杆菌KH2合成γ-聚谷氨酸的抑制机制

Inhibitory mechanism of glutamate waste liquid for poly-γ-glutamic acid production by Bacillus subtilis KH2

摘要:

谷氨酸废液是谷氨酸生产过程中的废弃物，具有低pH、高铵根、高硫酸根等特点。废液中含有谷氨酸，可作为原料用于生产γ-聚谷氨酸(poly-γ-glutamic acid, γ-PGA)，从而实现废液的资源化利用。目的 针对谷氨酸废液对γ-PGA合成存在抑制作用的问题，利用枯草芽孢杆菌(Bacillus subtilis) KH2进行发酵合成γ-PGA并评估谷氨酸废液对该合成反应的抑制情况。方法 通过转录组学比较，挖掘γ-PGA合成通路的关键基因，分析抑制因子，并利用关键基因的过表达与敲除技术，明确抑制因子，进而进行发酵验证。结果 谷氨酸废液作为底物发酵生产γ-PGA时呈现出显著的抑制效应。利用转录组学技术共筛选出1 819个显著差异基因，其中952个显著上调，867个显著下调。在原始发酵和谷氨酸废液发酵过程中，B. subtilis KH2中10个参与γ-PGA合成途径的基因(alsS、pgsA、gltT、budA、fumC、ptsG、racE、opuAB、acoC、rocG)转录水平发生明显变化。将其中8个下调表达的基因(alsS、pgsA、gltT、budA、fumC、ptsG、racE、opuAB)进行过表达并进行发酵验证后，γ-PGA产量分别提升了91.20%、120.77%、137.50%、36.44%、40.85%、104.58%、65.67%、69.72%；pgsA、gltT、ptsG、racE、opuAB基因的过表达使谷氨酸利用率分别提升了11.57%、35.53%、12.83%、21.43%、14.80%。alsS、budA、fumC的过表达对提升谷氨酸利用率效果不明显。2个上调表达基因(acoC、rocG)的敲除对γ-PGA生产和谷氨酸利用影响不大。结论 在废液发酵过程中，ptsG、gltT、racE、pgsA、fumC等基因的下调对底物利用、谷氨酸构型转换与聚合、TCA循环等产生显著影响，从而降低了γ-PGA的合成效率。本研究初步揭示了谷氨酸废液对γ-PGA合成的抑制机制，为利用工业废液生产高附加值生物聚合物提供了一种可持续的生物技术方法。

Abstract:

Glutamate waste liquid is the waste produced in the production process of glutamic acid, with low pH, high ammonium, and high sulfate. The waste liquid contains glutamic acid and can be used as a raw material to produce poly-γ-glutamic acid (γ-PGA), achieving the recycling of waste liquid.Objective To investigate the inhibitory effect of glutamate waste liquid on γ-PGA synthesis, we used Bacillus subtilis KH2 to synthesize γ-PGA and evaluated the inhibitory effect of glutamate waste liquid on the synthesis of γ-PGA.Methods Comparative transcriptomics was employed to excavate the key genes and inhibitory factors involved in γ-PGA synthesis, and key gene overexpression and knockout were conducted to identify the inhibitory factors. Fermentation experiments were then performed for verification.Results The glutamate waste liquid as the substrate for production of γ-PGA by fermentation showed significant inhibitory effects. A total of 1 819 significantly differentially expressed genes were identified, including 952 genes with significantly up-regulated expression and 867 genes with significantly down-regulated expression. The transcript levels of 10 genes (alsS, pgsA, gltT, budA, fumC, ptsG, racE, opuAB, acoC, and rocG) involved in γ-PGA synthesis of B. subtilis KH2 changed significantly during primary fermentation and glutamate waste liquid fermentation. Eight down-regulated genes (alsS, pgsA, gltT, budA, fumC, ptsG, racE, and opuAB) were overexpressed, which increased the production of γ-PGA by 91.20%, 120.77%, 137.50%, 36.44%, 40.85%, 104.58%, 65.67%, and 69.72%, respectively. The overexpression of pgsA, gltT, ptsG, racE, and opuAB increased glutamic acid utilization by 11.57%, 35.53%, 12.83%, 21.43%, and 14.80%, respectively. The overexpression of alsS, budA, and fumC had no obvious improving effect on the utilization of glutamic acid. The knockout of two up-regulated genes (acoC and rocG) had little effect on γ-PGA production and glutamic acid utilization.Conclusion The downregulation of ptsG, gltT, racE, pgsA, and fumC in waste liquid fermentation has significant effects on substrate utilization, glutamic acid configuration conversion and polymerization, and TCA cycle, which reduces the synthesis efficiency of γ-PGA. This study reveals the inhibitory mechanism of glutamate waste liquid in γ-PGA synthesis and provides a sustainable biotechnology for the production of value-added biopolymers from industrial waste liquid.