Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Vasco de Quiroga 4871, 05348, Mexico City, Mexico.
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Col. Chamilpa, 62210, Cuernavaca, MOR, Mexico.
Microb Cell Fact. 2022 Sep 7;21(1):183. doi: 10.1186/s12934-022-01906-1.
Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose.
The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L.
The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.
分批培养模式是工业生物工艺的标准培养技术。然而，大多数早期的细胞和工艺开发都是在分批培养中进行的，这可能会影响表达系统的初始选择。细胞工程可以为高通量筛选和宿主选择提供替代分批培养的方法。我们之前报道了一个大肠杆菌菌株文库，这些菌株具有单个和多个参与葡萄糖转运的基因缺失。与野生型（W3110）相比，突变株的葡萄糖摄取、生长和有氧乙酸盐生成率较低。因此，当在分批培养中培养时，这些突变体的表现可能与在分批培养中培养的 W3110 相似。为了验证这一假设，我们在微生物反应器中使用半补充培养基评估了绿色荧光蛋白（GFP）在分批培养中的组成型表达，该培养基补充有 10 或 20 g/L 葡萄糖+0.4 g 酵母提取物/g 葡萄糖。
在分批培养中培养的突变株显示出快速生长阶段（生长速率在 0.40 和 0.60 h 之间），随后是缓慢生长阶段（生长速率在 0.05 和 0.15 h 之间），类似于典型的分批补料培养。缓慢生长阶段很可能是由于关键氨基酸耗尽所致。三个突变株达到了最高的 GFP 荧光。特别是，一个名为 WHIC（ΔptsHIcrr、ΔmglABC）的突变株的 GFP 荧光达到了 W3110 的 14 倍以上。在 2 L 生物反应器中以 100 g/L 葡萄糖+50 g/L 酵母提取物的方式在分批培养中培养 WHIC 菌株。这些培养物与 W3110 的指数补料分批培养进行了比较，保持了 WHIC 的相同的缓慢生长（0.05 h），并且使用了与 WHIC 培养物相同的葡萄糖和酵母提取物总量。WHIC 菌株产生了约 450 mg/L GFP，而 W3110 仅产生了 220 mg/L GFP。
细胞工程和高通量筛选的结合允许选择一个在分批培养中模拟补料分批培养行为的特定突变体。此外，在分批和补料分批方案下，WHIC 菌株产生的 GFP 量都大大高于 W3110。因此，我们的结果为加速生物工艺开发提供了一项有价值的技术。
