代谢工程改造马克斯克鲁维酵母生产l-乳酸

Metabolic engineering of Kluyveromyces marxianus for l-lactic acid production

摘要:

l-乳酸作为一种重要的有机酸，在食品、医药和生物塑料等领域广泛应用。传统的l-乳酸生产主要依赖乳酸菌，但由于乳酸菌对底物要求高且对酸性环境的耐受性差，导致其工业化应用面临一定挑战。相比之下，马克斯克鲁维酵母(Kluyveromyces marxianus)因其快速生长、耐高温、底物利用广泛等优势，成为了乳酸生产的潜在宿主。为了优化马克斯克鲁维酵母的l-乳酸生产能力，本研究对其进行了代谢工程改造。首先，筛选并表征了高效的乳酸脱氢酶(lactic dehydrogenase, LDH)，然后利用CRISPR基因编辑技术在不同基因位点过表达LDH并敲除编码丙酮酸脱羧酶的基因PDC1，构建了菌株LA2.1，其l-乳酸产量达到72.55 g/L。通过筛选并组合耐酸靶点，进一步提升了菌株的耐酸性和乳酸产量。最终，通过工艺优化，菌株LA4.3发酵72 h积累了112.41 g/L的l-乳酸，得率为84.00%。在降低CaCO₃使用量的条件下，l-乳酸产量达到101.50 g/L，发酵结束后的pH值为3.52。本研究为l-乳酸的高效生产提供了理论基础和技术支持。

Abstract:

l-lactic acid is a vital organic acid with broad applications in food, pharmaceutical, and bioplastic industries. The conventional production of l-lactic acid predominantly relies on lactic acid bacteria, which are limited in the industrial application due to their strict substrate requirements and poor tolerance to acidic environments. Kluyveromyces marxianus has gained attention as a potential host for lactic acid production due to its rapid growth, acid tolerance, and ability to utilize inexpensive substrates. In this study, we aimed to enhance the lactic acid production capacity of K. marxianus through metabolic engineering and process optimization. We first overexpressed the lactate dehydrogenase (LDH) gene and employed CRISPR to knock out the pyruvate decarboxylase gene (PDC1), thus obtaining the engineered strain LA2.1, which achieved the l-lactic acid production of 72.55 g/L. By screening and combining acid tolerance targets, we improved the acid tolerance and lactic acid production of this strain. Through process optimization, the strain LA4.3 achieved the l-lactic acid production of 112.41 g/L and a yield of 84.00% within 72 h of fermentation. Additionally, in the case of reducing the calcium carbonate dosage, the l-lactic acid production reached 101.50 g/L, with pH 3.52 after fermentation. These findings highlight the potential of K. marxianus as a promising host for efficient l-lactic acid production and provide a solid theoretical and technical foundation for the industrial application of K. marxianus in bio-based chemical production.