高效Rubisco羧化活性筛选体系的设计与构建

Design and construction of an efficient selection system for Rubisco’s carboxylation

【背景】广泛存在于植物、藻类及其他自养微生物中的核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)是卡尔文循环中固定CO2的关键酶及限速酶,在生物质合成和全球碳循环中扮演着重要角色。鉴于Rubisco的重要性以及极低的固碳羧化活性,对Rubisco的筛选进化研究具有重要意义。【目的】构建一种适用于筛选高效Rubisco羧化活性的筛选体系。【方法】分析现有筛选体系对Rubisco羧化活性筛选压力缺失的原因,设计构建适用于高效Rubisco羧化活性的筛选体系,以BWLac产乳酸菌株为宿主,在含有5%CO2的N2环境下厌氧培养,比较不同羧化活性Rubisco对细胞生长的影响,通过HPLC及LCMS检测总乳酸及固定CO2生成乳酸的产量评估筛选体系。【结果】通过终端代谢产物乳酸产生下拉力,以及甘油代谢产生的剩余还原力需要平衡消耗掉的设计靶点,增强Prk和Rubisco的固碳支路代谢通量,加强RuBP对细胞的毒性抑制作用,使细胞生长与Rubisco活性有效偶联,构建高通量筛选办法。在新构建的筛选体系中,Rubisco失活突变体BWLac/197不能生长,Rubisco和Prk双失活突变体BWLac/197-2021生长未受影响,菌落大小约1.58 cm。粗酶液羧化活性相差2倍以上的RBC1和7002,其细胞生长在筛选条件下受不同程度抑制,菌落大小分别有1.06 cm和0.65 cm。通过检测乳酸产量及甘油消耗量对筛选体系验证评估,RBC1消耗甘油1.39 g/L,产乳酸2.82 g/L,其中来自于NaH13CO3的标记乳酸含量为18.05μmol/L,比7002的相应检测结果高1.3-1.6倍,检测结果与筛选体系设计原理相符,即乳酸产量越高,甘油消耗越多,Rubisco羧化活性越高,细胞生长得越好。【结论】成功设计构建了高效Rubisco羧化活性筛选体系,为进化或探索更高羧化活性Rubisco提供有效的高通量筛选办法。

[Background] Ribulose-1,5-bisphosphate caboxylase/oxygenase(Rubisco) is a key enzyme and the rate-limiting enzyme of CO2 fixation in the Calvin cycle, and widespread in plants, algae and other autotrophic microorganisms, playing an important role in biomass synthesis and the global carbon cycle. In view of the importance and the extremely low carbon-fixation activity of Rubisco, it is of great significance to study the selection evolution of Rubisco. [Objective] This study aims to construct a selection system suitable for screening efficient Rubisco carboxylation. [Methods] The reason of the absence of selection pressure on Rubisco carboxylation in the existing system was analyzed, then the new selection system applicable for high efficiency Rubisco carboxylation was designed and constructed. The lactate-producing strain BWLac was used as the host, the growth of Rubisco expression strains with different carboxylation activities were compared under the anaerobic culture containing 5% CO2, HPLC and LCMS were used to detect total lactic acid production and the yield of lactic acid from fixed CO2 for the evaluation of new constructed selection system. [Results] The design principle was considered to base on the pull-down force generated by the terminal metabolite lactic acid and the balance of the residual NADH generated by glycerol metabolism. These two parts could enhance the metabolic flux of the carbon-fixing branch of Prk and Rubisco, which could strengthen the inhibition of the toxicity of RuBP, coupling cell growth effectively with Rubisco activity to construct a high-throughput screening approach. In the newly constructed selection system, the Rubisco inactivated mutant BWLac/197 could not grow, while the growth of Rubisco and Prk double inactivated mutant BWLac/197-2021 was not affected with the colony size was about 1.58 cm. RBC1 and 7002, which carboxylation activities were detected to be more than 2 times different, the cell growth was inhibited to varying degrees under the screening conditions, and the colony size was 1.06 cm and 0.65 cm, respectively. The lactic acid production and glycerol consumption were also tested to evaluate the effectiveness of the selection system. RBC1 consumed 1.39 g/L of glycerol, produced 2.82 g/L of lactic acid, and labeled lactic acid content of 18.05 μmol/L, which was 1.3-1.6 times higher than the corresponding test result of 7002. The detection results were consistent with the design principle of the selection system, the higher the lactic acid production, the more glycerol consumption, the higher the Rubisco carboxylation, the better the cells grow. [Conclusion] An efficient selection system for Rubisco carboxylation was successfully designed and constructed to provide an effective high-throughput screening method for the evolution or exploration of Rubisco with higher carboxylation activity.