Engineering a carbon source-responsive promoter for improved biosynthesis in the non-conventional yeast Kluyveromyces marxianus

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Shane Bassett, Nancy A. Da Silva
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引用次数: 0

Abstract

Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast Kluyveromyces marxianus, by fusing carbon-source responsive elements of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter cores. Two hybrids, PIT350 and PIN450, were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of PIN450 increased TAL more than 50% relative to the native NC1 promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the Penicillium griseofulvum 6-methylsalicylic acid synthase (6-MSAS) under the control of PIN450 resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used PIN450 to express the Pseudomonas savastanoi IaaM and IaaH proteins and the Salvia pomifera sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for K. marxianus, allowing greater control over heterologous protein expression and improved production of toxic metabolites.

在非常规酵母 Kluyveromyces marxianus 中设计碳源响应型启动子以提高生物合成能力
许多理想的生物基化学品对微生物细胞工厂有不同程度的毒性,这使得工业级生物制造更具挑战性。众所周知,分离微生物生长和生产阶段有利于提高有毒产品的产量。在这里,我们通过将本地 ICL1 启动子的碳源响应元件与强大的 S. cerevisiae TDH3 或本地 NC1 启动子核心融合,开发出一种新型合成碳响应启动子,用于快速生长、耐受压力的酵母 Kluyveromyces marxianus。两个杂交种 PIT350 和 PIN450 通过 EGFP 荧光进行了验证,并分别在基于葡萄糖和乳糖的培养基中表现出卓越的强度、生长过程中的部分抑制和晚期激活。在 PIN450 的控制下表达非洲菊 2-吡喃酮合成酶(2-PS)以合成多酮三乙酸内酯(TAL),与原生 NC1 启动子相比,TAL 增加了 50%以上,额外的启动子工程进一步将试管培养中的 TAL 滴度提高到 1.39 克/升。在 PIN450 的控制下,表达青霉 6-甲基水杨酸合成酶(6-MSAS)使 6-MSA 滴度增加了 6.6 倍,达到 1.09 克/升,细胞生长也同时增加了 1.5 倍。最后,我们利用 PIN450 表达了 Pseudomonas savastanoi IaaM 和 IaaH 蛋白以及丹参桧烯合成酶蛋白,分别提高了对酵母有剧毒的辅助激素吲哚-3-乙酸和单萜桧烯的产量。碳响应启动子的开发增加了 K. marxianus 的合成生物学工具箱和可用的代谢工程策略,从而能够更好地控制异源蛋白的表达,并提高有毒代谢物的产量。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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