Increased Biomass Yield of Lactococcus lactis by Reduced Overconsumption of Amino Acids and Increased Catalytic Activities of Enzymes

Steady state cultivation and multidimensional data analysis (metabolic fluxes, absolute proteome, and transcriptome) are used to identify parameters that control the increase in biomass yield of Lactococcus lactis from 0.10 to 0.12 C-mol C-mol−1 with an increase in specific growth rate by 5 times from 0.1 to 0.5 h−1. Reorganization of amino acid consumption was expressed by the inactivation of the arginine deiminase pathway at a specific growth rate of 0.35 h−1 followed by reduced over-consumption of pyruvate directed amino acids (asparagine, serine, threonine, alanine and cysteine) until almost all consumed amino acids were used only for protein synthesis at maximal specific growth rate. This balanced growth was characterized by a high glycolytic flux carrying up to 87% of the carbon flow and only amino acids that relate to nucleotide synthesis (glutamine, serine and asparagine) were consumed in higher amounts than required for cellular protein synthesis. Changes in the proteome were minor (mainly increase in the translation apparatus). Instead, the apparent catalytic activities of enzymes and ribosomes increased by 3.5 times (0.1 vs 0.5 h−1). The apparent catalytic activities of glycolytic enzymes and ribosomal proteins were seen to follow this regulation pattern while those of enzymes involved in nucleotide metabolism increased more than the specific growth rate (over 5.5 times). Nucleotide synthesis formed the most abundant biomonomer synthetic pathway in the cells with an expenditure of 6% from the total ATP required for biosynthesis. Due to the increase in apparent catalytic activity, ribosome translation was more efficient at higher growth rates as evidenced by a decrease of protein to mRNA ratios. All these effects resulted in a 30% decrease of calculated ATP spilling (0.1 vs 0.5 h−1). Our results show that bioprocesses can be made more efficient (using a balanced metabolism) by varying the growth conditions.

本研究采用稳态培养（steady state cultivation）与多维数据分析方法，结合代谢通量（metabolic fluxes）、绝对蛋白质组（absolute proteome）与转录组（transcriptome）的多组学数据，筛选可调控乳酸乳球菌（Lactococcus lactis）生物量产率的关键参数：当菌株比生长速率（specific growth rate）从0.1 h⁻¹提升至0.5 h⁻¹（增幅达5倍）时，其生物量产率从0.10 C-mol C-mol⁻¹增至0.12 C-mol C-mol⁻¹。当比生长速率达到0.35 h⁻¹时，精氨酸脱亚胺酶途径（arginine deiminase pathway）被抑制，这一现象反映了氨基酸消耗模式的重塑；后续丙酮酸关联型氨基酸（天冬酰胺、丝氨酸、苏氨酸、丙氨酸与半胱氨酸）的过度消耗得到缓解，在最大比生长速率下，几乎所有摄入的氨基酸均仅用于蛋白质合成。这种平衡生长状态的特征为：糖酵解通量（glycolytic flux）极高，可承载高达87%的细胞碳流；且仅与核苷酸合成相关的氨基酸（谷氨酰胺、丝氨酸与天冬酰胺）的摄入量超出了细胞蛋白质合成的需求。蛋白质组的整体变化幅度较小，主要体现为翻译装置（translation apparatus）的丰度提升；与之相反，酶与核糖体的表观催化活性提升了3.5倍（比生长速率从0.1 h⁻¹升至0.5 h⁻¹时）。糖酵解酶与核糖体蛋白的表观催化活性均遵循这一调控规律，而核苷酸代谢相关酶的表观催化活性增幅则超过比生长速率的增幅（达5.5倍以上）。核苷酸合成是细胞内最主要的生物单体合成途径，其消耗的ATP占生物合成总ATP需求的6%。由于表观催化活性的提升，核糖体翻译在更高比生长速率下效率更高，这一点可通过蛋白质与mRNA的比值降低得到佐证。上述所有变化最终使计算得到的ATP泄漏（ATP spilling）量降低了30%（比生长速率从0.1 h⁻¹升至0.5 h⁻¹时）。本研究结果表明，通过调控生长条件优化代谢平衡，可有效提升生物工艺的运行效率。