Minnaar Letitia, den Haan Riaan
Department of Biotechnology, University of the Western Cape, Bellville, South Africa.
Appl Microbiol Biotechnol. 2023 Nov;107(22):7013-7028. doi: 10.1007/s00253-023-12729-4. Epub 2023 Sep 9.
Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.
酿酒酵母作为使用联合生物加工（CBP）方法生产纤维素生物乙醇的潜在宿主备受关注，这归因于其高产乙醇滴度、异源蛋白生产能力以及对各种工业相关压力的耐受性。由于酿酒酵母驯化菌株中异源蛋白的分泌水平通常较低，天然分离株可能提供更多样化的遗传背景以改善异源蛋白分泌，同时对工艺压力也表现出更强的稳健性。在本研究中，评估了天然和工业酿酒酵母菌株分泌一组核心纤维素酶（CBH1、CBH2、EG2和BGL1）的潜力，这些纤维素酶由使用CRISPR/Cas9工具整合的基因编码。与亲本菌株相比，高水平的异源蛋白生产与最大生长速率降低以及整体菌株稳健性的轻微变化有关。与参考工业菌株MH1000_BECC相比，天然分离株衍生物YI13_BECC和YI59_BECC分别在高培养温度和存在乙酸的情况下对异源纤维素酶表现出卓越的分泌能力。这些菌株还对几种发酵相关和分泌压力表现出多重耐受性。在限氧条件下于结晶纤维素上培养这些菌株，乙醇浓度在4 - 4.5 g/L范围内，相当于120小时后理论最大乙醇产量的35 - 40%，且无需添加外源酶。因此，本研究突出了这些天然分离株作为CBP生物乙醇生产底盘生物的潜力。要点：• 与工艺相关的发酵压力影响异源蛋白生产。• 转化体产生高达4.5 g/L乙醇，约为CBP中理论产量的40%。• CRISPR/Cas9可用于在天然酿酒酵母分离株中整合基因。
