Biomass Energy Technology Research Centre, Key Laboratory of Development and Application of Rural Renewable Energy (Ministry of Agriculture and Rural Affairs), Biogas Institute of Ministry of Agriculturegrid.464196.8 and Rural Affairs, Chengdu, People's Republic of China.
Graduate School of Chinese Academy of Agricultural Science, Beijing, People's Republic of China.
Appl Environ Microbiol. 2022 Feb 8;88(3):e0216121. doi: 10.1128/AEM.02161-21. Epub 2021 Nov 24.
Zymomonas mobilis (Z. mobilis) is a potential candidate strain for consolidated bioprocessing (CBP) in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion that is affected by cell wall peptidoglycan is also not well understood. Here, we constructed several penicillin-binding protein (PBP)-deficient strains derived from Z. mobilis S192 to perturb the cell wall peptidoglycan network and then investigated the effects of peptidoglycan on the endoglucanase secretion. The results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBP mutant strains, notably, Δ1089/0959 (4.09-fold) and Δ0959 (5.76-fold) in comparison to parent strains. For PBP-deficient strains, the growth performance was not significantly inhibited, but cell morphology was altered. In addition, enhanced antibiotic sensitivity and reduced inhibitor tolerance were also detected in our study. The concentration of intracellular soluble peptidoglycan was increased, especially for single-gene deletion. Outer membrane permeability of PBP-deficient strains was also improved, notably, Δ1089/0959 (1.14-fold) and Δ0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our findings indicated that PBP-deficient Z. mobilis was capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance, and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. Cell wall peptidoglycan has the function to maintain cell robustness and acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacteria. Herein, we perturbed the peptidoglycan synthesis network via knocking out PBPs (, , ) to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study could lay the foundation for understanding the regulatory network of cell wall synthesis and guide the construction of CBP strains in Z. mobilis.
运动发酵单胞菌（Zymomonas mobilis，Z. mobilis）是木质纤维素生物炼制中整合生物加工（Consolidated Bioprocessing，CBP）的潜在候选菌株。然而，纤维素酶的低水平分泌限制了这一 CBP 过程，并且细胞壁肽聚糖影响的蛋白质分泌机制也尚未得到很好的理解。在这里，我们构建了几株源自运动发酵单胞菌 S192 的青霉素结合蛋白（Penicillin-Binding Protein，PBP）缺陷株，以扰乱细胞壁肽聚糖网络，然后研究了肽聚糖对内切葡聚糖酶分泌的影响。结果表明，在 PBP 突变株中，重组内切葡聚糖酶的胞外产量显著提高，特别是Δ1089/0959（4.09 倍）和Δ0959（5.76 倍）与亲本菌株相比。对于 PBP 缺陷株，生长性能没有受到明显抑制，但细胞形态发生了改变。此外，我们的研究还检测到增强的抗生素敏感性和降低的抑制剂耐受性。细胞内可溶性肽聚糖浓度增加，特别是在单个基因缺失时。PBP 缺陷株的外膜通透性也得到了改善，特别是Δ1089/0959（1.14 倍）和Δ0959（1.07 倍），这可能解释了内切葡聚糖酶的胞外分泌增加。我们的研究结果表明，通过扰乱细胞壁肽聚糖，PBP 缺陷的运动发酵单胞菌能够增加内切葡聚糖酶的胞外分泌，这将为未来运动发酵单胞菌中 CBP 技术的发展提供基础。细胞壁肽聚糖具有维持细胞稳健性的功能，并作为细胞质中重组蛋白分泌到细胞外空间的屏障，在运动发酵单胞菌和其他细菌中都是如此。在此，我们通过敲除 PBPs（，，）来干扰肽聚糖合成网络，以增强运动发酵单胞菌 S912 中的重组内切葡聚糖酶的胞外分泌。这项研究可以为理解细胞壁合成的调控网络奠定基础，并指导运动发酵单胞菌中 CBP 菌株的构建。
