Modified recombinant yeast manufacturing process for efficient commercial large-scale production of enzymes.

“生物催化剂有限公司是一家位于英国卡迪夫的中小企业。利用微生物发酵技术，我们生产高价值的酶，销售到以下市场应用：（i）用作生产药物中间体的加工助剂（ii）用作生产食品配料的加工助剂（iii）用于生命科学应用中的DNA加工。我们有几个微生物平台，我们使用它们来生产包含细菌和酵母的重组酶。当我们为新市场开发战略性酶产品或为客户项目开发新型酶产品时，我们会通过生产设计流程对酶进行认真评估。这是为了最大限度地提高可能性，如果我们可以在实验室规模生产酶，那么我们将能够扩大到大规模商业上可行的发酵和下游工艺，以满足成本目标。有些酶比细菌更适合在酵母中表达，我们目前默认的酵母生产平台是巴斯德毕赤酵母。在毕赤酵母中，在学术和商业规模上最广泛使用的表达系统涉及使用甲醇作为酶（蛋白质）高水平表达的诱导剂。在生产环境中大规模使用甲醇充满了经济、健康和安全以及潜在的环境限制。因此，生物催化剂目前仅限于甲醇诱导的毕赤酵母发酵至500 L的规模。我们目前正在扩大我们的生产能力超过2年的计划，以8,000升。装备具有在此发酵规模下使用甲醇的能力的这种设施是极其昂贵的。目前，我们有3种酶产品在毕赤酵母中用甲醇诱导大规模生产，因此我们有义务将这项业务出口到欧洲的合作伙伴公司。因此，该项目旨在开发一种具有成本效益的，高产的无甲醇表达系统，用于酵母中的商业生产规模_巴斯德毕赤酵母_，以使我们的业务在英国扩大这一领域。"

"Biocatalysts Ltd are an SME located in Cardiff UK. Using microbial fermentation technology, we manufacture high-value enzymes that are sold into the following market applications: (i) used as processing aids in the manufacture of pharmaceutical intermediates (ii) used as processing aids in the manufacture of food ingredients (iii) used for DNA processing in Life Science applications.We have several microbial platforms that we use to manufacture recombinant enzymes incorporating both bacteria and yeasts. When we develop a strategic enzyme product for a new market or a novel enzyme product for a customer project we perform a diligent assessment of the enzyme with our Design for Manufacture process. This is aimed at maximising the probability that if we can make the enzyme at laboratory scale then we will be able to scale-up to large scale commercially viable fermentations and downstream processes to meet cost targets. Some enzymes are more suitable for expression in yeast than bacteria and our current default yeast manufacturing platform is in _Pichia pastoris_. The most widely used expression system in _Pichia_ at both the academic and commercial scale involves the use of Methanol as an inducer for the high-level expression of the enzyme (protein). Large-scale use of methanol in a manufacturing context is fraught with economic, health and safety and potential environmental constraints. Consequently Biocatalysts is currently limited to methanol-induced Pichia fermentations to a scale of 500 L. We are currently expanding our manufacturing capacity over a 2 year plan to 8,000 L. It is prohibitively expensive to equip such a facility with the capacity to work with methanol at this fermentation scale. Currently we have 3 enzyme products produced at large-scale in _Pichia_ with methanol induction, so consequently we are obliged to export this business to a partner company in Europe. Therefore, this project is aimed at developing a cost effective, high yielding methanol-free expression system for commercial manufacturing scale in the yeast _Pichia pastoris_ to allow our business to expand in this area in the UK."