2023, Vol. 39中国科学院微生物研究所、中国微生物学会主办
文章信息
- 汤晓玲, 陈静祥, 柳志强, 郑裕国
- TANG Xiaoling, CHEN Jingxiang, LIU Zhiqiang, ZHENG Yuguo
- 基于工业环境扰动的微生物适应性进化
- Adaptive evolution of microorganisms based on industrial environmental perturbations
- 生物工程学报, 2023, 39(3): 993-1008
- Chinese Journal of Biotechnology, 2023, 39(3): 993-1008
- 10.13345/j.cjb.220781
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文章历史
- Received: September 28, 2022
- Accepted: December 9, 2022
引用本文
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随着现代生物技术和合成生物学的发展，设计最优合成途径、重构生化合成网络、创制新元件及与途径、细胞适配，驱动微生物细胞工厂的构建，成为变革物质生产模式的重要方向。与传统化学路线相比，通过构建高效微生物细胞工厂及开发绿色生产关键技术，可有效解决代谢物质流和能量流的定向控制问题、降低生产成本、减少三废排放，对发展促进工业经济的可再生原料路线、推进物质财富的绿色增长具有重大意义。
然而，高强度工业环境下抗逆性差、工程放大效率低成为了限制了微生物细胞工厂生产性能的关键因素。工业生产中高渗透压、高底物/产物浓度胁迫以及高酸碱物质浓度等因素，影响并扰动细胞繁衍、生产能力及环境耐受力，降低细胞生产水平。因此，改善并解决细胞在工业环境扰动下的生产性能问题对提高工业环境下细胞工厂生产效率具有重要意义[1-2]。适应性进化是目前受到广泛关注的微生物改良技术，能够在较短的时间内，根据人工施加的定向选择压力，获得适应特定条件的耐受工业环境的有效细胞，从而提高微生物生产性能[3-4]。
1 微生物适应性进化应用领域微生物适应性进化(microbial adaptive evolution)是针对细胞的工业环境不适应性，人为模拟自然选择，优化微生物对应激源的耐受性，累积有益突变应对选择压力，获得驯化菌株的生物工程技术(图 1)[5-6]。在复杂的工业环境中，适应性进化可通过优化生长速率、提升细胞对高浓度底物或产物的耐受性、平衡细胞生长与生产等策略促进细胞的生产性能，逐步成为构建高生产强度的微生物细胞工厂的有效工具[7-8](图 2)。
传统的定向进化主要聚焦单个蛋白质序列，通过随机突变，筛选获得性能显著提高的突变体，实现分子水平的模拟进化[9]。定向进化为改进蛋白质结构与功能提供了有效手段，但单一基因或单一途径的改造难以实现多尺度性能的提升。相比较而言，微生物的适应性进化通过基因组的全局进化，获得与环境相适应且具有鲁棒性能的工程菌株[10-11]。该类进化可以在单一宿主细胞中实现多个不同性状的同时改造，涉及对数个、数十个基因的突变或修饰，从而应对多重胁迫响应[12-14]。
2 微生物适应性进化关键技术微生物适应性进化过程中，通过人工施加定向的选择压力，使微生物沿着所需的方向进化，从而获得目标性状[15-16]。但是少数突变表型在长期适应性进化后才可凸显，且需在稳定期前不断稀释转接维持细胞内外压力恒定，操作过程冗杂，并行性差，在适应性过程中容易增加交叉污染的风险[17-18]。随着合成生物学技术的发展，适应性进化模型的构建和应用、微流控技术以及生物传感器的发展成为有效解决这一瓶颈的重要工具。在多组学分析的辅助下，全面系统地解析适应性微生物的突变遗传机制，挖掘基因调控的有效方式，进一步促进微生物细胞工厂的生产性能。
2.1 适应性进化模型的构建及应用基于微生物本身特性构建的适应性进化模型为筛选并获得特定表型的突变体提供重要指导。如构建耐盐适应性进化模型增加了模型酵母细胞大小、生产性能[19-20]。构建酒精耐受适应性进化模型增加了酿酒酵母细胞壁的稳定性，进而提升其对高浓度酒精的耐受性[21]。
Isenring等[22]通过计算机辅助建立连续聚合酶发酵(polymerase fermentation, PolyFermS)模型探究适应性进化对结肠微生物群的适用性，发现控制肠道微生物群的应用环境可允许单一菌株在高度可控的环境中进行适应性进化，从而提高植物乳杆菌(Lactobacillus plantarum) NZ3400的进化效率。Zhang等[23]通过拟合线性和非线性参数，建立地衣芽孢杆菌(Bacillus licheniformis)生长耦合适应性进化模型。进一步通过微液滴集成化的流动型反应器分析建立基于荧光信号强度的高通量筛选模式，分离获得生长状态优良的突变株，并考察其对淀粉酶的分泌效率。结果表明，60 h后α-淀粉酶活性为645.9 U/mL，为同等条件下原始菌株的1.9倍。在添加α-淀粉酶的培养基中突变体生长能力与酶活性呈正相关，验证了该模型的可靠性和指导意义。
2.2 微流控技术在微生物适应性进化中的应用微流控技术是一种用于控制极微量液体的新型技术，把细胞培养、实验处理、成像、检测等步骤高度集成到芯片上，实现高通量快分析和筛选的目的。微流控技术应用于微生物适应性进化，可避免无义突变干扰，显著提升突变体的筛选效率。
微生物可在微流控技术产生的高度均一液滴中进行生化反应，改变液滴硬度。如乳酸菌产生的乳酸与碳酸钙反应释放钙离子，继而与海藻酸钠发生交联，引发液滴凝胶化[24]。郭肖杰[25]设计了一种基于微滴硬度分选高产乳酸菌的微流控芯片。产酸效率高的菌株凝胶速度快，继而对硬度大的微滴进行扩大培养，筛选突变库，获得高产乳酸的进化菌株。
近几年，研究人员开发全自动高通量微生物液滴培养仪(microbial microdroplet culture system, MMC)系统也逐渐用于研究微生物的适应性进化，为高效选育工业微生物提供了便利。Li等[26]以大肠杆菌(Escherichia coli) MeSV2.2为出发菌，在含肉汤的摇瓶中培养72 h后将菌体接种到悬液于新鲜培养基，培养5 h后再次转接至MMC继代培养30 h，MMC中的液滴重新接种于肉汤培养基，如此循环反复，进行18 d的适应性进化，分选获得的进化菌株的生长速度比亲本提高18.5%和17.3%。研究证明了MMC在提升微生物生长速率方面的潜力，为筛选工业模式下的适应性菌株提供了一种有效且可靠的方法。
此外，流式细胞仪能够纯化细胞群体，基于荧光物质的表达情况进一步分析优势微生物。如基于l-缬氨酸与荧光蛋白(enhance yellow fluorescent protein, eYFP)耦合的原理，借助流式细胞仪的分选技术，选择荧光最强的细胞，从而分离出l-缬氨酸产量提高的菌株[27-28]。微流控技术与流式细胞仪的高效相结合，将提升适应性进化菌株的分选速率，开拓工业微生物新发展。
2.3 生物传感器在微生物适应性进化中的应用在工业生产中，微生物会对不断变化的环境产生敏感性。生物传感器将敏感物质浓度转换为可测量的电信号，利用分析装置检测并记录分析物的生理生化变化，为监测和量化内源性环境信号、阐明生物过程提供了重要手段。高灵敏度和高保真度的生物传感器与高效筛选技术的有效结合是适应性进化成功的关键之一[29-30](图 3)。
Della等[31]建立了基于LysR家族转录调控因子生物传感器。将来源于谷氨酸棒状杆菌(Corynebacterium glutamicum)的LysR家族转录调控因子LysG，与胞内诱导物赖氨酸、精氨酸或组氨酸结合，进而结合到lysE的启动子区域，激活碱性氨基酸转运蛋白编码基因lysE的表达，维持胞内碱性氨基酸的稳态。进一步通过对LysG的改造，提高其对赖氨酸的诱导响应值，成功应用于C. glutamicum赖氨酸回补途径中关键酶磷酸烯醇式丙酮酸羧化酶的高通量筛选，从而获得l-赖氨酸生产效率显著提高的突变体。
Stella等[32]通过基于转录调节因子Lrp建立的生物传感器控制生长调节基因pfkA (编码磷酸果糖激酶)或hisD (编码组氨醇脱氢酶)。根据Lrp生物传感器对蛋氨酸、亮氨酸，异亮氨酸和缬氨酸的灵敏度不同，将谷氨酸棒杆菌ATCC 13032 WT的生长与细胞内支链氨基酸的浓度相偶联，分离出两种能够提高氨基酸产量的工程菌株。随后对两种突变菌株PbrnF-pfkA和PbrnF-hisD进行重复分批适应性进化。在5次重复培养后，突变菌株中PbrnF启动子区域发生变化(插入442个G)、Lrp中的氨基酸被替换(A替换C)及pfkA基因发生复制(复制率为18%)。转录调节因子指导下的生物传感器简化了突变工作，成功地将进化应用于改善化学品的生产。
2.4 多组学分析在微生物适应性进化中的应用通过基因组、转录组、蛋白组和代谢组的多组学分析有利于解析微生物在进化过程中的关键突变点和遗传机制[33-34]。基于激增的组学数据，从多层面进行比较和进化分析，有利于探究细胞的适应性进化历程及对工业环境的适应机制。Kim等[35]建立了基因组规模代谢网络模型(genome-scale metabolic model, GEM)。在该模型的指导下，解析了适应性进化后大肠杆菌代谢途径中磷酸转乙酰酶pta和乳酸脱氢酶ldhD的敲除对丁酸盐生成的影响，结果表明两个酶的双敲除使丁酸盐产量提高了3.4倍，比生长速率提高4.8倍，从而指导该宿主细胞的进一步进化。Purdy等[36]利用聚球藻菌(Synechococcus elongatus)的基因组尺度代谢模型，将大量非天然NADH合成途径引入进化菌株的代谢网络，通过促进生产途径上游的NADH生成来提高生物燃料醇的合成。结果表明，进化后工程菌的酒精滴度增加71%，异丁醇增加43%，2-甲基-1-丁醇的合成效率也显著提升。代谢模型的应用为改造进化菌株提供了重要思路。
3 适应性进化提高微生物性能 3.1 优化微生物生长速率微生物代谢过程中因受到环境压力，发生基因扰动，干扰其生理机能，导致生长的不适应，降低生长速率，这种不适的生理状态可通过中枢代谢的适应性进化得到恢复[37]。Wannier等[38]将大肠杆菌中321个终止密码子UAG替换为UAA，敲除I类肽释放因子1 (prfA编码的RF1)，获得大肠杆菌C321.ΔA。RF1的缺失导致菌株出现“适合度缺陷”，生长严重受损，故在最低浓度葡萄糖培养基中对C321.ΔA实施1 100代的适应性进化。进化种群的测序结果表明随着传代次数递增，所有菌株在培养基中的适应性有所改善，生长速率明显增长(> 40%)，重新编码的进化群体中发生了选择性突变，独立重新编码的谱系突变中摆脱了“适合度缺陷”，重组菌株实现了自适应性进化。
Cassler等[39]对毕赤酵母(Pichia pastoris) CBS7435衍生菌的中心碳代谢进行了重组，并进行连续传代适应性进化。进化菌株的甲醇利用率是初始菌的1.5倍，生长速率由0.008 h–1提高至0.015 h–1。全基因组测序表明，进化菌株的prk基因(编码烟酸单核苷酸腺苷转移酶)中存在单核苷酸多态性(single nucleotide polymorphisms, SNPs)，导致烟酸单核苷酸腺苷转移酶发生突变，prk活性降低，增强代谢途径中的ATP可用水平，改善进化菌的自养生长。对突变菌株进行第二次适应性进化，使用CRISPR/Cas9介导的反向基因工程进一步研究发现，适应性进化增加过氧化物酶体结合的PEX5比例，加速蛋白转运，使进化菌株的生长速率再次提高至0.018 h–1，γ-氨基丁酸的产量提升约20%。基于进化和代谢工程的结合优化工程菌株的生长速率，促进生物合成，为筛选新兴的工业化菌株奠定了基础。
Pfeifer等[40]对谷氨酸棒杆菌ATCC 13032进行适应性进化，筛选可在低葡萄糖培养基上生长速率提升的菌株。历经100代后，分离菌株(UBw和UBm)在低糖培养基上的生长速率提高了26%。基因组测序可知，关键基因pyk (丙酮酸激酶)、fruK (1-磷酸果糖激酶)和corA (Mg2+转运蛋白酶)发生突变，pyk、fruK的突变使葡萄糖消耗率和ptsG表达增加，致使菌株在低糖情况下加速生长，corA突变促进进化株在缺镁条件下生长。
适应性进化也被用于提升微藻类的生长速率。Perrineau等[41]对莱茵衣藻(Chlamydomonas reinhardtii) CC-503 cw92 mt+进行适应性进化，经1 880传代培养后，进化种群的细胞增长率比前体细胞提高35%。基因组测序发现，在进化细胞中有1 937个多肽DNA区域，其中149个SNPs导致氨基酸取代，参与蛋白质合成、细胞分裂和增殖的基因上调，从而促进细胞增长，以此说明了适应性进化在改善藻类基因组方面的潜在能力，为研究藻类品系提供了策略，对推进真核微生物的研究有巨大帮助。适应性进化提高微生物生长速率的典型实例见表 1。
| Strain | Evolution strategy | Result | Reference |
| E. coli K-12 MG1655(C321.ΔA) | Continuous batch culture for 1 100 generations | Compared with the initial strain, the growth rate of the “ΔRF1” strain was increased by > 40%, the “fitness defect” was eliminated, and the growth was recovered | [38] |
| P. pastoris (Komagataella phaffii) CBS7435 derivative | The central carbon metabolism pathways were reconstructed and adaptive evolution experiments were performed | The gene PEX5 encoding the peroxisomal input receptor was mutated, and the growth rate of the evolved strain increased by 1.25 times compared with the initial strain | [39] |
| C. glutamicum ATCC13032 | 100 generations of adaptive evolution on medium with low concentration of glucose | The growth rate of the screened strains UBw and UBm on medium with low sugar content was increased by 26% | [40] |
| C. reinhardtii CC-503 cw92 mt+ | 1 880 generations of subcultures | The population cell growth rate of the evolved strain was 35% higher than that of the initial cells | [41] |
| Engineered autotrophic E. coli | 250 chemostat generations of cultures in media with restricted carbon source | The efficiency of CO2 utilization for biomass was improved | [42] |
在微生物发酵过程中，往往受到底物、产物抑制，继而降低细胞生产能力。此外，某些产物会毒害菌体，终产物的累积亦会对代谢途径中的关键基因产生负反馈抑制。对受底物、产物反馈抑制的菌株实施适应性进化以改善微生物表型、增强耐受性和提高产量也是微生物细胞工厂构建的一个重要目标。
丙酸(propionic acid, PA)是重要的C-3有机酸，然而丙酸的累积对工业丙酸杆菌的生产有强烈抑制作用。Cavero-Olguin等[43]对丙酸杆菌(Propionibacterium acidipropionici)进行了适应性进化。初始菌株在含10、20、30 g/L丙酸的培养基中迭代培养。最终在含有20 g/L丙酸的初始培养基中筛选出优势菌株，其生长速度是野生菌的1.4倍，丙酸产量是初始菌的2倍。以16S rRNA基因作为对照，使用Real timequantitative PCR (RT-qPCR)检测treY (麦芽低聚糖海藻糖合成酶)和otsA (海藻糖6-磷酸合成酶)基因的表达，结果发现treY基因在适应性进化中的表达得到了强化。Matson等[44]为解除产物抑制，对生产乙酸异丁酯(isobutyl acetate, IBA)的大肠杆菌JCL260进行耐IBA的适应性进化。在适应性进化耐受模型的指导下，经37轮耐压筛选获得22株耐高浓度IBA突变体，产量提升了3.2倍。
乙醇是酿酒酵母发酵后期的主要抑制剂，高浓度乙醇抑制菌体活性，影响菌体寿命。Zhang等[45]对模型酿酒酵母(Saccharomyces cerevisiae) YF10-5进行液氮冻融，并置于乙醇胁迫下筛选分离获得YF10-5突变株。研究发现，突变株在发酵前期和后期的海藻糖积累量分别比出发菌株高42.9%和20.5%。通过qPCR分析YF10-5突变株的转录反应可知，与应激反应相关的基因HSP26、HSP30及HSP104 (热休克蛋白酶)、海藻糖合成基因TPS1、调节乙醇代谢和参与胞内海藻糖累积基因ADH1、HXK1和PFK1表达上调，表明高浓度海藻糖的响应胁迫促使菌株耐受高渗环境，致使乙醇生产能力达到13.4% (质量体积分数)，比亲本提升16%，相当于工业理论生产率的93.95%。
通常情况下，适应性进化与物理或化学手段相结合共同促进菌株进化。常温常压等离子诱变(atmospheric room temperature plasma, ARTP)是常用的辅助工具。Jiang等[46]以酿酒酵母菌株SyBE SC1402为初始菌株，利用ARTP与H2O2交叉诱导的适应性进化策略，筛选出3个突出突变体(AX13、AX14和AX15)，虾青素产量分别提高至59.36、63.22、65.93 mg/L，均比起始菌株提高了4倍。进一步探究发现，适应性进化干扰了菌株的基因组，增加了葡萄糖到类胡萝卜素的碳通量，从而提高β-胡萝卜素到虾青素的转化效率。
适应性进化与生物诱变的有效结合可显著提高微生物突变率，为筛选高产化合物的工业菌株及构建微生物细胞工厂提供了思路(表 2)。
| Expected phenotype | Strain | Adaptive process | Result | Reference |
| Improve acid tolerance | P. acidipropionici DSMZ 4900 | The propionic acid concentration in the medium was gradiently increased to induce the cells’ tolerance | The evolved strain could grow in the medium containing 20 g/L propionic acid, the growth rate was 1.4 times that of the wild type, and the propionic acid yield was increased by 2-folds | [43] |
| Improve substrate tolerance | E. coli JCL260 | 37 rounds of cultivation on medium with high-concentration of IBA | The IBA producing capacity of the evolved was increased by 3.2-folds and the evolution mechanism was elucidated | [44] |
| Improve ethanol tolerance | S. cerevisiae YF10-5 | Continuous cultivation under the condition of high concentration ethanol stress | The ethanol content of the evolved strain was increased by 16%, and the growth rate was much higher than that of the original strain | [45] |
| Improve l-serine tolerance | E. coli lacking l-serine degradationpathways | 45 days of evolution of the strain in media with increased l-serine concentration | The capacibity of the evolved strains grew at high l-serine concentration was improved and the productivity of l-serine production was increased (reached 37 g/L) | [47] |
| Improve substrate tolerance | E. coli JCL260 | Different inhibitory concentrations of glucosamine-6-phosphate were added to the strain medium | The cell membrane synthesis was promoted and isobutanol-tolerated mutants were obtained | [48] |
在工业生产环境中，细胞的生长与生产不平衡，导致代谢流分散是限制其生产能力的另一重要因素。通过细胞的适应性进化，解除生产阻滞，最大限度地平衡生长与生产，是提升微生物的工业生产效率的重要手段[49-51]。
Prell等[52]基于代谢工程手段对谷氨酸棒杆菌GluA T0进行改造，敲除l-谷氨酸脱氢酶基因gdh，平衡细胞生长和戊二酸的生产。之后将指数期细胞密度最高的悬液转移到含1 mmol/L IPTG的新鲜培养基中进行多次传代培养，利用纳米孔测序技术和Illumina基因组测序揭示进化菌株的突变机制。结果表明，编码琥珀酸半醛脱氢酶GabD的基因发生点突变，促进了琥珀酸半醛转化为琥珀酸，进化使细胞的生长速率由0.10 h−1提高至0.17 h–1。另一方面，gdh缺失导致从α-酮戊二酸到谷氨酸的碳流量减少，从而积累了α-酮戊二酸，促进戊二酸的生产，产量达22.7 g/L，时空产率为0.35 g/(L·h)。
NAD+再生对细胞体内能量的产生和关键细胞过程的调节至关重要，使靶向生物合成途径成为主要的NAD+再生途径，有利于细胞生长与生产[53-55]。Pontrelli等[56]以大肠杆菌JCL166为初始菌，过表达fdh编码的甲酸脱氢酶，增加NADH再生，促进1-丁醇的合成。同时删除磷酸乙酰转移酶GPAM，提高乙酰辅酶A利用率，使生长不受抑制。对改造后的菌株进行适应性进化，于厌氧发酵环境中连续转接稀释30次，结果表明菌株的生长和生产直接关联，能够在24 h生产600 mg/L 1-丁醇。对进化机制进行解析，发现编码鸟氨酸转氨甲酰酶的arcB突变基因参与了对厌氧条件下细胞生长的基因调控，在此基础上通过进一步进化，1-丁醇的产量达到2 g/L。
利用生物传感器快速监测产品输出与突变率，分析生长与生产的平衡度，在基因组测序的辅助下确定适应性进化中的新突变，可指导细胞的进化[57]。Yu等[58]设计大肠杆菌代谢型生物传感器感应细胞体内番茄红素浓度，以此监测大肠杆菌的适应性进化程度。通过使用较弱的启动子降低异柠檬酸脱氢酶(isocitrate dehydrogenase, IDH)的表达来调控ATP生成和番茄红素生物合成之间的代谢通量分布。经过432 h适应性进化，番茄红素产量提升了16倍。全基因组测序表明编码丙酮酸脱氢酶的aceE基因发生突变，催化丙酮酸的氧化脱羧，促进丙酮酸流向TCA循环，加快菌体生长，从而提升番茄红素产量。这些研究数据充分表明在生物传感器指导下的适应性进化能够维持代谢生长与生产间的平衡，激发工程菌株的生产潜力(表 3)。
| Strain | Screening strategy | Result | Reference |
| C. glutamicum (GluA T0) | Knockout of the l-glutamate dehydrogenase gene, together with adaptive evolution | The growth rate of the isolated mutant was increased to 0.17 h–1, the yield of glutaric acid was 22.7 g/L, and the reverse extraction yield was 99%, which was twice that of the parental strain | [52] |
| E. coli JCL166 | Improvement of NADH regeneration and acetyl-CoA utilization, together with adaptive evolution under anaerobic environment | The growth and production of the strain were positively correlated, and the yield of product 1-butanol reached 2 g/L | [56] |
| E. coli | Adaptive evolution based on biosensor-monitored lycopene concentration | The lycopene production was increased by 16-folds and growth was accelerated | [58] |
| E. coli B8/pED | Metabolic engineering coupled with adaptive evolution under high concentration of tryptophan | The yields of l-Trp and deoxyviolet were increased by 100 times and 2.5 times respectively | [59] |
| E. coli BW25113 | Mutation of d-lactate dehydrogenase coupled with adaptive evolution under high concentration of isobutanol | The adaptively evolved strain produced 21.2 g/L isobutanol within 99 hours, which was 76% of the theoretical maximum | [60] |
天然微生物存在对工业条件耐受性差、生成性能差等不足，难以直接用于工业生产。利用合适的胁迫压力进行适应性进化，激活微生物沉默基因簇，克服高温、高盐等高渗胁迫，筛选与工业环境相适应的菌株，提高细胞生产效率，促进细胞工厂产业化。
任喜东等[61]对生产ε-聚赖氨酸(ε-PL)的链霉菌株(Streptomyces diastatochromogenes) T17实施酸性适应性进化，筛选得到3株进化菌株(S. diastatochromogenes AE44、S. diastatochromogenes AE51和S. diastatochromogenes AE56)。初始菌株正常生长的pH值为6.5，进化后可承受的pH为3.2，胞内ATP含量和H+-ATPase活性高于原始菌，进化后的ε-PL生产菌可应对酸胁迫的生理响应，为ε-PL在工业强酸环境中的合成奠定重要基础。
Huang等[62]以酿酒酵母S288C的衍生菌株BY4741为亲本菌株，逐步增加培养温度，在42 ℃条件下获得14个进化株。全基因组测序表明进化株产生的SWI/SNF复合体(一种较大的多亚基、ATP依赖性核小体重塑复合体)和F型ATPase中存在外显子突变，揭示了高温耐受性原因。在Hog1、Ras-cAMP和Rho1-Pkc1 (三者均为胞外信号调节激酶)参与的信号转导网络中发现多种突变，致使细胞可以通过改变现有的应激调节机制实现Htg+突变(该基因与高温生长相关)。该研究增加了对酵母细胞在异常高温下的生存和生长遗传机制的了解。
在酵母发酵乙醇工业中，高温对稳定产量有显著作用[63]。一般在35 ℃以下，酿酒酵母发酵途径中的酶活性低，终产物途径受阻，而耐热酵母可解除该缺陷。Serafim等[64]从酿酒酵母AMY12 (PE-2)和AMY35 (SA-1)出发，通过适应性进化获得耐高温菌株。与亲本相比，两株进化菌在40 ℃条件下的酶活分别提高63%和61%，糖醇转化率提高至44%和41%。Caspeta等[65]以酿酒酵母S288C为起始株，在(39.5±0.5) ℃条件下传代培养342 d，在40 ℃发酵条件下获得乙醇产量为70 g/L的菌株TTY23。随着乙醇浓度的增加，菌株TTY23的产量再次提高15.4%。转录组分析表明，PMA1 (编码P型质子输出转运蛋白酶)下调、HSP30 (H+-ATP酶的负调节因子)上调，线粒体活性提升，使菌株TTY23可在40 ℃下生长。
Dhar等[66]在酿酒酵母培养基中添加0.5 mol/L NaCl提升细胞对高盐的适应力。细胞生长分裂300代，得到3株进化后的菌株，生长速率均优于亲本，细胞大小比亲本株系增大约8%– 12%。通过微阵列分析发现，有82个表达基因变化，62个调节基因变化，群体测序表明CTT1 (过氧化氢酶)、MSN4 (应激反应性转录激活因子)和HLR1 (参与调节细胞壁组成和渗透胁迫)这3个应激反应基因的表达量增加，进而增加微生物对高盐环境的耐受性。通过适应性进化筛选所需生理代谢特征的突变菌株，揭示了变异的分子遗传机制[67]，对高效开发酵母菌株的工业应用潜力具有重要意义。
Shim等[68]使用氧化应激作为选择压力对酿酒酵母BY4741进行适应性进化。结果表明，CTT1编码的过氧化氢酶可增强酵母对H2O2的防御能力，在不同浓度H2O2中进行筛选获得突变体YAG115，对H2O2的耐受性最强，持续暴露于100 mol/L H2O2环境中仍可生长。在此基础上，提升了该菌株对目标产物β-石竹烯的生成能力。Reyes等[69]综合利用代谢工程手段进行细胞的适应性进化实验，进一步通过氧化应激策略刺激代谢途径分支靶点的关键酶，增加新突变体，耦合细胞的生长与生产，并通过微流控技术分离出进化后的优势酵母菌，对β-石竹烯的产量进一步提升。
适应性进化提升微生物耐受性的典型案例见表 4。
| Strain | Screening strategy | Result | Reference |
| S. diastatochromogenes T17 | Adaptive evolution under pH stress | The obtained three evolved strains, AE44, AE4451 and AE4456 exhibited higher ability to synthesize ε-polylysine (ε-PL) | [61] |
| S. cerevisiae BY4741 | Adaptive evolution under high temperature stress | Thermophilic yeasts were screened and the grow rate at 42 ℃ was improved | [62] |
| Brazilian industrial strains of S. cerevisiae (PE-2 and SA-1) | Adaptive evolution under high temperature stress | The biomass of the evolved strains PE-2 and SA-1 was increased by 63% and 61% at 40 ℃, respectively | [64] |
| S. cerevisiae S288C | Adaptive evolution under high temperature and high concentrations of substrates stress | The evolved strain TTY23 could tolerate high concentrations of ethanol at 40 ℃, with a 15.4% increase in yield | [65] |
| S. cerevisiae | 300 generations of cultivation in medium supplemented with 0.5 mol/L NaCl | The growth rate of the evolved strain was increased by 8%–12%, with variable and larger cell morphology | [66] |
| S. cerevisiae BY4741 | Adaptive evolution under oxidative stress | The yield of target product β-caryophyllene was increased and the growth was improved when the cells were exposured to 100 mol/L hydrogen peroxide | [68] |
适应性进化在增加微生物耐受性、提升生长速率和平衡生长与生产的同时，可激活“沉默代谢途径”，促进底物利用，降低工业成本。在工业发酵领域，提高微生物发酵能力的同时，也会产生其他特征性化合物，如酿酒过程中芳香化合物和蛋氨酸发酵中高丝氨酸衍生物的生成，这些高附加值产物同样具有重要的经济效益[70]。但是构建非天然化合物的高效细胞工厂往往因受关键节点掣肘而难度大，代谢途径的提升空间有限[71]。利用非理性设计的适应性进化策略能够有效提升细胞生产性能[72]。如Choi等[73]使用l-异亮氨酸的结构类似物α-氨基丁酸(α-aminobutyric acid, α-AB)作为抗性筛选标记物，从代谢节点入手，解除异亮氨酸对苏氨酸脱氢酶的负反馈抑制作用，通过ARTP使l-异亮氨酸的产量提高了1.61倍。
另一方面，微生物的代谢网络错综复杂，适应性进化可作为有效工具挖掘工程菌的未知功能性基因，扩展生物信息库。如Tai等[74]通过适应性进化鉴别出大肠杆菌中的功能性非磷酸化基因簇，为代谢改造提供了新思路。当需要在特定环境中选择期望表型时，适应性进化可以取代理性设计，追溯随机突变的源途经，根据一定数量且效果良好的突变基因簇，将适应性识别的等位基因外源导入菌株，设计所需表型，诱导获得特定的细胞性状[75]。
4 总结与展望微生物细胞工厂的应用潜力很大程度取决于其在工业环境下的生产能力。微生物难以耐受高强度工业环境，抗逆性差，成为了限制生产性能的关键因素。综合运用微流控、生物传感器等技术，在特定工业环境扰动下，对微生物进行适应性进化，优化生长速率，提升对高浓度底物/产物的耐受性，强化对工业环境压力如高温、高酸碱、高盐等的适应性，平衡细胞生长与生产能力，成为拓展微生物在高强度环境下的应用提供了重要策略。
随着合成生物学等技术的快速发展，系统研究微生物适应性进化机制，建立理论和技术的一体化研究系统，将奠定微生物细胞工厂工业环境下高效运行的工程基础。(1) 通过高强度、逆性发酵环境下微生物代谢组、转录组、蛋白组研究，将解析其高产和对抗工业环境的分子机制，阐明生产和耐受之间相互影响的机理，获得与生产和耐受耦联的标记性代谢物或蛋白及与发酵环境变化、细胞膜通透性、细胞活性的对应关系，从而可对细胞与环境的耦合度进行快速评价，指导适应性进化的开展。(2) 通过发展高效基因组编辑工具，包括大片段DNA组装技术、多重基因组自动改造技术等，将实现微生物基因组高效多位点模块插入和功能修饰。使用启动子、非翻译区、核糖体结合位点、转录终止子等DNA调控元件，可时空调控维持细胞耐受生长，同时保持细胞的高产性能。(3) 通过发展基于响应特定环境因素的转录调控因子及其作用启动子的生物传感器，可建立工业高产菌种库的高通量筛选方法，提升适应性进化效率，改进微生物特性，获得与环境耦合度好的进化菌株。(4) 通过利用转座子突变、质粒编码的基因组文库、全局转录机器改造、DNA复制酶修正功能改编等技术，可全局性扰动微生物基因组，模拟工业生产环境，设定高产物浓度胁迫、高温、高渗透压等环境压力，全面提高微生物对工业环境压力的耐受性，最终构建能够耐受极端生产环境、生产强度高的高效微生物细胞工厂，从而有力推动合成生物产业的健康发展，为构建“高效、清洁、低碳、循环”的绿色制造体系，促进社会的可持续、绿色发展作出积极贡献。
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