A bio-based production of chemical building blocks from renewable, sustainable and non-food substrates is one key element to fight climate crisis. Lactic acid, one such chemical building block is currently produced from first generation feedstocks such as glucose and sucrose, both requiring land and water resources. In this study we aimed for lactic acid production from methanol by utilizing Komagataella phaffii as a production platform. Methanol, a single carbon source has potential as a sustainable substrate as technology allows (electro)chemical hydrogenation of CO2 for methanol production. Here we show that expression of the Lactiplantibacillus plantarum derived lactate dehydrogenase leads to L-lactic acid production in Komagataella phaffii, however, production resulted in low titers and cells subsequently consumed lactic acid again. Gene expression analysis of the methanol-utilizing genes AOX1, FDH1 and DAS2 showed that the presence of lactic acid downregulates transcription of the aforementioned genes, thereby repressing the methanol-utilizing pathway. For activation of the methanol-utilizing pathway in the presence of lactic acid, we constructed strains deficient in transcriptional repressors Nrg1, Mig1-1, and Mig1-2 as well as strains with overrepresentation of transcriptional activators Mxr1 and Mit1. While loss of transcriptional repressors had no significant impact on lactic acid production, overexpression of both transcriptional activators, MXR1 and MIT1, increased lactic acid titers from 4 g L−1 to 17 g L−1 in bioreactor cultivations.

中文翻译：

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转录激活因子 Mxr1 和 Mit1 的过表达增强了 Komagataellaphaffii 中甲醇上乳酸的产生


利用可再生、可持续和非食品基材生产化学基础原料是应对气候危机的关键因素之一。乳酸就是这样一种化学基础材料，目前由葡萄糖和蔗糖等第一代原料生产，这两种原料都需要土地和水资源。在这项研究中，我们旨在通过利用 Komagataella phaffii 作为生产平台，从甲醇中生产乳酸。甲醇是一种单一碳源，具有作为可持续基材的潜力，因为该技术允许对 CO2 进行（电）化学加氢以生产甲醇。在这里，我们表明植物乳植杆菌衍生的乳酸脱氢酶的表达导致 Komagataella phaffii 产生 L-乳酸，然而，产生导致低滴度，细胞随后再次消耗乳酸。甲醇利用基因 AOX1 、 FDH1 和 DAS2 的基因表达分析表明，乳酸的存在下调了上述基因的转录，从而抑制了甲醇利用途径。为了在乳酸存在下激活甲醇利用途径，我们构建了缺乏转录抑制因子 Nrg1 、 Mig1-1 和 Mig1-2 的菌株，以及过表达转录激活因子 Mxr1 和 Mit1 的菌株。虽然转录抑制因子的缺失对乳酸的产生没有显著影响，但转录激活因子 MXR1 和 MIT1 的过表达使生物反应器培养中的乳酸滴度从 4 g L-1 增加到 17 g L-1。