School of Biological Sciences, College of Science, Georgia Institute of Technology, Atlanta, Georgia.
Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia.
Biotechnol Bioeng. 2020 Jun;117(6):1696-1709. doi: 10.1002/bit.27318. Epub 2020 Mar 25.
A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.
生物制造的成本效益面临的一个障碍是工程遗传元件（如质粒）的不稳定性。当真菌双核体由于细胞分裂过程中的核分离而返回到亲本物种时，整个基因组水平也会出现不稳定性。在这里，我们通过将酿酒酵母-毕赤酵母双核体包裹在藻酸盐基质中，展示了可以限制细胞分裂并保持其扩展代谢能力。作为纤维素乙醇生产的替代方法，我们测试了这些细胞进行葡萄糖和木糖乙醇发酵的能力，研究了与浮游融合体以及由这些物种混合组成的浮游和包裹细胞培养物相关的底物利用、倍性和细胞活力。与浮游对照相比，包裹双核体的葡萄糖和木糖消耗以及乙醇生产更高。没有发生同时共发酵；相反，包裹双核体中葡萄糖和木糖分解代谢的顺序和动力学与两种物种一起包裹的培养物相似。在重复的分批培养循环中，与浮游融合体相比，包裹的酿酒酵母-毕赤酵母融合体的基因组稳定性显著增加。封装还增加了用于标记每种物种的抗生素抗性质粒的稳定性，并在混合培养物中保持了酿酒酵母和毕赤酵母细胞的固定比例。我们的数据表明，细胞在细胞外基质中被包裹如何限制细胞分裂，从而保存从基因组到质粒再到混合群体的实体的稳定性和生物活性，其中每一个对于成本效益高的生物制造都是必不可少的。
