Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus
Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus
复制标题
DOI：
10.1002/bit.26976
复制
发表时间：
2019-07-01
影响因子：
3.8
通讯作者：
Da Silva, Nancy A.
中科院分区：
文献类型：
--
作者：
McTaggart, Tami L.;Bever, Danielle;Da Silva, Nancy A.
马氏克鲁维酵母具有生长速度快、TCA循环通量高、耐低pH和耐高温等特点，是一种很有前途的非常规生物化工生产酵母。与酿酒酵母不同，马克氏克雷伯氏菌在低成本的基质上生长，其细胞密度等于或超过葡萄糖中的密度，这有利于利用木质纤维生物质(木糖)、生物燃料生产废物(甘油)和乳清(乳糖)。以三乙酸内酯(TAL)为产物，对马氏链霉菌合成聚酮类化合物进行了评价。在CEN/ARS载体上表达了2-吡喃酮合酶(2-PS)，并研究了温度、碳源和培养策略对TAL水平的影响。在37℃的1%木糖介质中获得了最高滴度，在41℃和43℃时有相当的滴度。引入高稳定性的2-PS突变体和启动子替代使滴度增加了四倍。以多拷贝PKD1为基础的2-PS表达使TAL效价进一步提高了5倍。将最好的质粒、启动子和菌株组合在一起，在3ml试管培养中，这个非工程菌株的TAL效价为1.24g/L，产量为0.0295 molTAL/molC。在代谢工程或补料分批培养之前，相对于其他宿主(在葡萄糖上)，这是一个极好的滴度和产量(木糖)，并展示了这种快速生长和耐热的酵母品种在聚酮生产中的前景。
Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37 degrees C, with substantial titers at 41 and 43 degrees C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.