DataSheet1_Enabling growth-decoupled Komagataella phaffii recombinant protein production based on the methanol-free PDH promoter.pdf

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of Komagataella phaffii (Pichia pastoris) can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using K. phaffii has often been driven either by the methanol induced AOX1 promoter (PAOX1) and/or the constitutive GAP promoter (PGAP). In the last years, strong efforts have been focused on developing novel expression systems that expand the toolbox variety of K. phaffii to efficiently produce diverse proteins that requires different strategies. In this work, a study was conducted towards the development of methanol-free expression system based on a heat-shock gene promoter (PDH) using glycerol as sole carbon source. Using this promoter, the recombinant expression is strongly induced in carbon-starving conditions. The classical PGAP was used as a benchmark, taking for both strains the lipase B from Candida antarctica (CalB) as model protein. Titer of CalB expressed under PDH outperformed PGAP controlled expression in shake-flask cultivations when using a slow-release continuous feeding technology, confirming that PDH is induced under pseudo-starving conditions. This increase was also confirmed in fed-batch cultivations. Several optimization rounds were carried out for PDH under different feeding and osmolarity conditions. In all of them the PDH controlled process outperformed the PGAP one in regard to CalB titer. The best PDH approach reached 3.6-fold more specific productivity than PGAP fed-batch at low μ. Compared to the optimum approach for PGAP-based process, the best PDH fed-batch strategy resulted in 2.3-fold higher titer, while the specific productivity was very similar. To summarize, PDH is an inducible promoter that exhibited a non-coupled growth regulation showing high performance, which provides a methanol-free additional solution to the usual growth-coupled systems for RPP. Thus, this novel system emerges as a potential alternative for K. phaffii RPP bioprocess and for revaluing crude glycerol, promoting the transition towards a circular economy.

当前向循环生物经济（circular bioeconomy）转型的进程，需要合理开发生物炼制厂（biorefineries），以可持续满足当下的各类需求。巴斯德毕赤酵母（Komagataella phaffii，旧称Pichia pastoris）的应用可应对这一挑战，因其能够以生物柴油工业副产物粗甘油（crude glycerol）作为唯一碳源，同时生产高、低附加值产物。利用巴斯德毕赤酵母进行重组蛋白生产（Recombinant Protein Production, RPP）时，通常采用甲醇诱导型AOX1启动子（PAOX1）和/或组成型GAP启动子（PGAP）驱动表达。近年来，研究人员已投入大量精力开发新型表达系统，以丰富巴斯德毕赤酵母的表达工具库，高效生产需要不同策略实现的各类蛋白质。本研究针对开发基于热休克基因启动子（heat-shock gene promoter, PDH）的无甲醇表达系统展开探索，该系统以甘油作为唯一碳源。该启动子可在碳饥饿条件下强力诱导重组蛋白表达。研究以经典的PGAP作为参照，且两种菌株均采用南极假丝酵母脂肪酶B（lipase B from Candida antarctica, CalB）作为模式蛋白。当采用缓释连续补料技术进行摇瓶培养（shake-flask cultivations）时，PDH驱动表达的CalB效价优于PGAP调控的表达体系，证实PDH可在伪饥饿条件下被诱导激活。这一结果在补料分批培养（fed-batch cultivations）中也得到了验证。研究团队针对PDH系统开展了多轮优化，分别调控不同的补料方式与渗透压条件。在所有优化场景中，PDH调控的工艺在CalB效价方面均优于PGAP体系。在低比生长速率（μ）的补料分批培养中，最优PDH工艺的比生产率（specific productivity）较PGAP体系高出3.6倍。相较于PGAP基工艺的最优方案，最优PDH补料分批策略的蛋白效价提升了2.3倍，而比生产率基本相当。综上，PDH是一种诱导型启动子，其表现出与生长解偶联的调控特性且性能优异，为常规的生长偶联型重组蛋白生产系统提供了一种无甲醇的替代方案。因此，该新型系统有望成为巴斯德毕赤酵母重组蛋白生产工艺以及粗甘油资源化利用的潜在替代方案，助力循环经济转型。