Suwannakham Supaporn, Yang Shang-Tian
Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210, USA.
Biotechnol Bioeng. 2005 Aug 5;91(3):325-37. doi: 10.1002/bit.20473.
Fed-batch fermentations of glucose by P. acidipropionici ATCC 4875 in free-cell suspension culture and immobilized in a fibrous-bed bioreactor (FBB) were studied. The latter produced a much higher propionic acid concentration (71.8 +/- 0.8 g/L vs. 52.2 +/- 1.1 g/L), indicating enhanced tolerance to propionic acid inhibition by cells adapted in the FBB. Compared to the free-cell fermentation, the FBB culture produced 20-59% more propionate (0.40-0.65 +/- 0.02 g/g vs. 0.41 +/- 0.02 g/g), 17% less acetate (0.10 +/- 0.01 g/g vs. 0.12 +/- 0.02 g/g), and 50% less succinate (0.09 +/- 0.02 g/g vs. 0.18 +/- 0.03 g/g) from glucose. The higher propionate production in the FBB was attributed to mutations in two key enzymes, oxaloacetate transcarboxylase and propionyl CoA: succinyl CoA transferase, leading to the production of propionic acid from pyruvate. Both showed higher specific activity and lower sensitivity to propionic acid inhibition in the mutant than in the wild type. In contrast, the activity of PEP carboxylase, which converts PEP directly to oxaloacetate and leads to the production of succinate from glucose, was generally lower in the mutant than in the wild type. For phosphotransacetylase and acetate kinase in the acetate formation pathway, however, there was no significant difference between the mutant and the wild type. In addition, the mutant had a striking change in its morphology. With a threefold increase in its length and approximately 24% decrease in its diameter, the mutant cell had an approximately 10% higher specific surface area that should have made the mutant more efficient in transporting substrates and metabolites across the cell membrane. A slightly lower membrane-bound ATPase activity found in the mutant also indicated that the mutant might have a more efficient proton pump to allow it to better tolerate propionic acid. In addition, the mutant had more longer-chain saturated fatty acids (C17:0) and less unsaturated fatty acids (C18:1), both of which could decrease membrane fluidity and might have contributed to the increased propionate tolerance. The enhanced propionic acid production from glucose by P. acidipropionici was thus attributed to both a high viable cell density maintained in the reactor and favorable mutations resulted from adaptation by cell immobilization in the FBB.
研究了嗜酸丙酸杆菌ATCC 4875在游离细胞悬浮培养中以及固定在纤维床生物反应器（FBB）中对葡萄糖进行的补料分批发酵。后者产生的丙酸浓度要高得多（71.8±0.8 g/L对52.2±1.1 g/L），这表明适应FBB的细胞对丙酸抑制的耐受性增强。与游离细胞发酵相比，FBB培养从葡萄糖产生的丙酸盐多20 - 59%（0.40 - 0.65±0.02 g/g对0.41±0.02 g/g），乙酸盐少17%（0.10±0.01 g/g对0.12±0.02 g/g），琥珀酸盐少50%（0.09±0.02 g/g对0.18±0.03 g/g）。FBB中较高的丙酸盐产量归因于两种关键酶，草酰乙酸转羧酶和丙酰辅酶A：琥珀酰辅酶A转移酶的突变，从而导致从丙酮酸产生丙酸。在突变体中，这两种酶均表现出比野生型更高的比活性以及对丙酸抑制更低的敏感性。相反，将磷酸烯醇式丙酮酸羧化酶直接将磷酸烯醇式丙酮酸转化为草酰乙酸并导致从葡萄糖产生琥珀酸盐的活性，在突变体中通常低于野生型。然而，对于乙酸形成途径中的磷酸转乙酰酶和乙酸激酶，突变体与野生型之间没有显著差异。此外，突变体的形态有显著变化。突变体细胞长度增加了三倍，直径大约减小了24%，其比表面积大约高10%，这应该使突变体在跨细胞膜转运底物和代谢物方面更高效。在突变体中发现的膜结合ATP酶活性略低也表明突变体可能有更高效的质子泵，使其能够更好地耐受丙酸。此外，突变体具有更多的长链饱和脂肪酸（C17:0）和更少的不饱和脂肪酸（C18:1），这两者都可以降低膜流动性，可能有助于提高对丙酸的耐受性。因此，嗜酸丙酸杆菌从葡萄糖中提高的丙酸产量归因于反应器中维持的高活细胞密度以及细胞固定在FBB中适应产生的有利突变。
