Auxenochlorella protothecoides adapted to grow efficiently in low phosphate conditions display altered lipid profiles. Auxenochlorella protothecoides adapted to grow efficiently in low phosphate conditions display altered lipid profiles

Renewable biodiesel produced by microalgae has great potential as a portable source of high-density energy that can replace certain traditional hydrocarbon sources. One limitation of scaling up algal cultivation to industrial levels is the availability of key macronutrients, particularly inorganic phosphorus (Pi), which is a finite and dwindling resource. Here, Auxenochlorella protothecoides was adapted to low Pi conditions through continuous cultivation in 100 times less Pi media for 285 days, or ~41 generations. The adapted populations demonstrated significantly higher growth rates than wild type (WT) cells in low Pi, with average maximum growth rates of 0.72 d-1 and 0.54 d-1, respectively in batch-culture experiments. Based on UPLC/MS analyses, the total lipid content of the adapting A. protothecoides populations showed a shift from their typical profile in nutrient replete media to the accumulation of non-phosphorus glycerolipids, with 306% MGDG and 189% SQDG, relative to WT at initial exposure, followed by a decline, and a steady increase until the final time point was reached. Transcriptome profiling by Illumina RNA-seq collected at 5 time points throughout the experiment, and results of the lipid analyses revealed a trend of increased transcript levels during the first ~11 generations of adaptation, followed by an overall decrease in gene expression after ~34 generations. The short-term changes in gene expression were associated with shifts in major metabolic pathways including carbon metabolism, oxidative phosphorylation, glycolysis, and gluconeogenesis. By comparison, certain transcripts showing decreased expression, reflected increased fatty acid turnover, and a stable decrease in photosynthesis-related gene expression. These results illustrate the utility of laboratory-directed evolution for the selection of microalgae populations with altered cultivation traits, revealing distinct phases of adaptation, based on expression profiles. The results further demonstrate the use of metabolic engineering to select a microalga variant after only ~40 generations of growth in low-phosphate conditions that utilizes Pi more efficiently for growth than its wild type parent population and produces 1.22 times more biomass in batch growth experiments. Overall design: mRNA levels for triplicate wild type cell populations (WT1, WT2, and WT3) compared to mRNA levels of triplicate cell populations grown in low phosphate media (LP1, LP4, and LP5) collected at four time points during adaptation over >41 generations.

由微藻制备的可再生生物柴油，作为可替代部分传统烃类能源的高密度便携能源载体，具备巨大应用潜力。将藻类规模化培养至工业级规模的核心瓶颈之一，在于关键大量营养素的供给不足，尤其是无机磷（Pi）——这是一种储量有限且日渐枯竭的资源。本研究通过在磷含量仅为原培养基1/100的培养基中连续培养285天（约41代），使原壳小球藻（Auxenochlorella protothecoides）适应低磷环境。经驯化的种群在低磷环境下的生长速率显著高于野生型（WT）细胞；批量培养实验显示，二者的平均最大生长速率分别为0.72 d⁻¹和0.54 d⁻¹。基于超高效液相色谱-质谱联用（UPLC/MS）分析结果，驯化过程中原壳小球藻种群的总脂质组成发生显著转变：从营养充足培养基中的典型谱型，转向非磷脂甘油酯的积累；相较于初始暴露时的野生型，其单半乳糖基二酰基甘油（MGDG）与硫代异鼠李糖甘油二酯（SQDG）的含量分别提升306%和189%，随后含量有所回落，并在抵达最终采样时间点前持续稳步上升。本研究在实验全程的5个时间点采用Illumina RNA测序（RNA-seq）开展转录组谱分析，结合脂质分析结果发现：在驯化的前约11代期间，转录本水平呈上升趋势，而在约34代后，基因表达整体出现下调。基因表达的短期变化与多条核心代谢通路的重塑密切相关，涵盖碳代谢、氧化磷酸化、糖酵解及糖异生通路。相较而言，部分表达下调的转录本对应脂肪酸周转效率的提升，同时与光合作用相关的基因表达持续稳定下调。上述结果证实了实验室定向进化技术在筛选具备定制化培养性状的微藻种群中的应用价值，并基于基因表达谱揭示了驯化过程的不同阶段。本研究进一步证明，通过在低磷环境中仅生长约40代，即可借助代谢工程手段筛选得到优化的微藻变体：该变体相较于野生型亲本种群，可更高效地利用无机磷进行生长，且批量培养实验中的生物量较亲本提升1.22倍。实验整体设计：将三组生物学重复的野生型细胞种群（WT1、WT2、WT3）的mRNA表达水平，与在低磷培养基中培养、且在超过41代的驯化过程中四个时间点采集的三组生物学重复细胞种群（LP1、LP4、LP5）的mRNA表达水平进行对比分析。