Zhao Jianzhi, Zhao Yuping, Wu Longhao, Yan Ning, Yang Shuo, Xu Lili, He Deyun, Li Hongxing, Bao Xiaoming
Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology, Shandong Academy of Sciences, 3501 Daxue Road, Jinan 250353, China.
Microorganisms. 2024 Jul 25;12(8):1526. doi: 10.3390/microorganisms12081526.
The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.
第二代生物乙醇的经济高效商业化生产需要能够完全且快速利用木质纤维素水解产物中所有糖类的发酵微生物。在本研究中，我们通过原生质体融合和代谢工程开发了一种重组菌株BLH510，以增强其在耐受木质纤维素水解产物中常见的各种抑制剂的同时，共发酵葡萄糖、木糖、纤维二糖和低聚木糖的能力。亲本菌株LF1和BLN26分别因其卓越的葡萄糖/木糖共发酵能力和抑制剂耐受性而被选中。融合菌株BLH510在限氧条件下表现出对混合糖的高效利用和高乙醇产量。在低接种量条件下，菌株BLH510能够在84小时内完全消耗培养基中的所有四种糖类。发酵产生了33.96 g/L乙醇，达到理论乙醇产量的84.3%。尽管存在具有挑战性的混合抑制剂，BLH510在发酵120小时后仍成功代谢了上述所有四种糖类，产生了约30 g/L乙醇，达到理论产量的83%。此外，菌株BLH510的细胞内海藻糖含量增加，特别是在存在混合抑制剂的条件下，细胞内海藻糖达到239.3 mg/g酵母生物量。这种升高的海藻糖含量有助于增强BLH510的胁迫耐受性。该研究还优化了原生质体制备和融合的条件，平衡了高制备效率和令人满意的再生效率。结果表明，BLH510是用于从木质纤维素生物质生产工业第二代生物乙醇的有前途的候选菌株，在具有挑战性的发酵条件下表现出改进的性能。我们的工作证明了结合原生质体融合和代谢工程来开发用于木质纤维素生物乙醇生产的优良菌株的潜力。这种方法也可以扩展到开发用于生产多种基于木质纤维素生物质的生化产品的强大微生物平台。
