Department of Integrated Applied Life Science, Integrated Graduate School of Medicine, Engineering, and Agricultural Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
Department of Integrated Applied Life Science, Integrated Graduate School of Medicine, Engineering, and Agricultural Sciences, University of Yamanashi, 4-4-37 Takeda, Kofu, Yamanashi 400-8510, Japan.
Biochem Biophys Res Commun. 2024 Nov 12;733:150704. doi: 10.1016/j.bbrc.2024.150704. Epub 2024 Sep 14.
Botryococcus braunii is a colonial alga recognized for its slow growth but high hydrocarbon accumulation. Although using genetic engineering to increase the growth rate and hydrocarbon yield of B. braunii is desirable, the presence of an extracellular matrix (ECM) significantly hinders the emergence of a homogeneous colony from a single DNA-transformed cell. Previously, we developed a method to isolate single cells without ECM from colonies. However, following the isolation of single cells, several months are required to regenerate colonies with a sufficient cell mass for subsequent analysis. To shorten the colony regeneration period, we investigated basal media and medium components, along with growth-promoting additives, in a series of single-factor experiments and optimized the concentrations of the medium constituents via response surface methodology (RSM). The results of the single-factor experiments revealed that the nitrogen source (a mixture of NaNO and NHNO), 1-naphthylacetic acid (NAA) and Fe(III)-citrate significantly increased the growth of B. braunii single cells into colonies. The optimal medium composition identified by RSM included 151.6 mg/L nitrogen source, 2.419 mg/L NAA and 15.3 mg/L Fe(III)-citrate. Verification experiments showed that the optimized medium resulted in a 1.75-fold increase in colony size compared with that of colonies grown in nonoptimized AF6 medium. This is the first report of the optimal medium composition for the regeneration of B. braunii colonies from single cells.
胶球藻是一种生长缓慢但具有高烃类积累能力的群居藻类。尽管利用基因工程提高胶球藻的生长速度和烃类产量是可取的，但由于存在细胞外基质（ECM），从单个 DNA 转化细胞中产生均匀的菌落仍然具有挑战性。以前，我们开发了一种从菌落中分离无 ECM 的单细胞的方法。然而，在分离单细胞后，需要几个月的时间才能再生出具有足够细胞质量的菌落，以便进行后续分析。为了缩短菌落再生周期，我们在一系列单因素实验中研究了基础培养基和培养基成分，以及生长促进添加剂，并通过响应面法（RSM）优化了培养基成分的浓度。单因素实验的结果表明，氮源（NaNO 和 NHNO 的混合物）、1-萘乙酸（NAA）和 Fe（III）-柠檬酸盐显著促进了胶球藻单细胞向菌落的生长。RSM 确定的最佳培养基组成为：氮源 151.6 mg/L、NAA 2.419 mg/L 和 Fe（III）-柠檬酸盐 15.3 mg/L。验证实验表明，与在非优化的 AF6 培养基中生长的菌落相比，优化后的培养基使菌落大小增加了 1.75 倍。这是首次报道从单细胞再生胶球藻菌落的最佳培养基组成。
