1. 天津大学 化工学院 教育部合成生物学前沿科学中心 系统生物工程教育部重点实验室，天津 300072;
2. 天津大学 天津化学化工协同创新中心，天津 300072
收稿日期：2020-09-24；接收日期：2021-02-01
基金项目：国家重点研发计划(No. 2018YFA0902100) 资助
摘要：石油烃污染物属于难降解混合物，生物修复已经成为石油烃污染环境的主要修复方法。文中简述了微生物对石油烃的间期适应过程和转运过程，并通过对部分典型石油烃成分的微生物降解机理和代谢路径的梳理和综述，阐释了石油烃生物降解过程中的菌株、基因、代谢路径等研究进展。此外，利用基因工程和代谢工程等手段，可对野生型石油烃降解菌进行改造，进一步提升其对石油烃污染环境的生物修复能力。最后，从石油烃降解菌的代谢途径改造、人工混菌体系的设计构建等角度，结合合成生物学和代谢工程的手段，提出了对石油烃降解的研究展望，以期提升对石油烃污染物的生物修复效果。
关键词：石油烃降解合成生物学代谢工程人工混菌体系
Advances in biodegradation of petroleum hydrocarbons
Hengchang Li1,2, Mingzhu Ding1,2
1. Key Laboratory of Systems Bioengineering, Ministry of Education, Frontier Science Center for Synthetic Biology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
2. Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China
Received: September 24, 2020; Accepted: February 1, 2021
Supported by: National Key Research and Development Program of China (No. 2018YFA0902100)
Corresponding author: Mingzhu Ding. Tel: +86-22-60973987; E-mail: mzding@tju.edu.cn.
Abstract: Petroleum hydrocarbon pollutants are difficult to be degraded, and bioremediation has received increasing attention for remediating the hydrocarbon polluted area. This review started by introducing the interphase adaptation and transport process of hydrocarbon by microbes. Subsequently, the advances made in the identification of hydrocarbon-degrading strains and genes as well as elucidation of metabolic pathways and underpinning mechanisms in the biodegradation of typical petroleum hydrocarbon pollutants were summarized. The capability of wild-type hydrocarbon degrading bacteria can be enhanced through genetic engineering and metabolic engineering. With the rapid development of synthetic biology, the bioremediation of hydrocarbon polluted area can be further improved by engineering the metabolic pathways of hydrocarbon-degrading microbes, or through design and construction of synthetic microbial consortia.
Keywords: petroleum hydrocarbon degradationsynthetic biologymetabolic engineeringsynthetic microbial consortia
生物修复是一种符合绿色发展理念的主要修复方式，并逐渐成为研究重点。生物修复石油烃污染环境大致可以分为以下3个过程，微生物对石油烃的间期适应过程、转运过程和降解过程。在生物修复石油烃污染环境的过程中，微生物首先会对石油烃进行间期适应过程，在此过程中微生物一般通过分泌表面活性剂和趋化运动两种方式增大对石油烃污染物的生物利用度[1-2]；然后，石油烃经过转运过程进入细胞内，主要方式有自由扩散、被动运输、主动运输和内吞作用[3]，且大部分石油烃的转运过程需要转运蛋白的参与；最后，石油烃在细胞内完成降解，石油烃分子一旦进入细胞内，即被加氧酶氧化为脂肪族醇，脂肪族醇依次转化为脂肪族酸和脂酰辅酶A等，随后经β-氧化降解(图 1)[4]。本文总结和梳理了生物修复石油烃污染环境的研究进展，并展望了该领域的研究方向。
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| 图 1 石油烃在微生物中的代谢示意图 Fig. 1 Overview of degradation of petroleum hydrocarbon in microorganisms. |
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1 微生物对石油烃的间期适应过程和转运过程1.1 微生物对石油烃的间期适应过程由于石油烃污染物在水中的溶解度很低，在生物修复石油烃污染环境的过程中，微生物首先会对石油烃进行间期适应过程。在间期适应过程中微生物可以通过分泌表面活性剂增大与石油烃的接触，还可以通过趋化运动靠近石油烃污染物[2]，从而达到提高石油烃污染物生物利用度的目的(图 2)。
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| 图 2 微生物对石油烃间期适应过程的两种方式 Fig. 2 Two modes of interphase adaptation of petroleum hydrocarbon by microorganisms. (A) Secretion of surfactants. (B) Chemotaxis of microorganisms. |
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一方面，在间期适应过程中，部分微生物可通过分泌表面活性剂降低黏度、增加流动性等使石油的采收率得到提高。有研究表明，铜绿假单胞菌Pseudomonas aeruginosa NCIM 5514在60 d内显著降低原油黏度，增加原油的流动性[5]。微生物还可以通过释放表面活性剂改变油水接触面的表面张力、增加细胞表面的疏水性，从而增大微生物对疏水性底物的接触与利用(图 2A)。有研究人员分离出了一种由不动杆菌Acinetobacter indicus M6产生的生物表面活性剂，该生物表面活性剂可以显著降低表面张力[6]。短小芽孢杆菌Bacillus pumilus 1529产生的生物表面活性剂有助于将有毒的菲转化为毒性较小的代谢物[7]。以上研究表明，微生物表面活性剂在提升生物修复的效果中发挥着重大作用。但是，目前看来，合适的工程菌和经济的下游工艺是微生物表面活性剂工业生产的限制条件。从分子生物学和合成生物学的角度出发，改造野生菌株、构建工程菌株以及优化下游工艺条件成为解决此问题的最佳策略。
另一方面，在间期适应过程中，石油烃降解菌(主要是一些带鞭毛和菌毛的细菌) 还可以有效感应进而通过趋化运动靠近石油烃污染物。有研究者通过建立数学模型定量分析了恶臭假单胞菌Pseudomonas putida G7对萘的趋化性，这表明了趋化性在萘降解过程中的重要作用[2]。细菌大多数借助鞭毛或菌毛趋化[8]。其中模式生物大肠杆菌具有最简单的趋化信号通路，研究最为广泛。其趋化机制为：(1) 细胞通过受体蛋白感应到底物的存在并发出信号；(2) 磷酸化水平发生变化；(3) 鞭毛的旋转方向决定细菌的运动方向。然而，细菌借助菌毛的趋化研究相对较少，特别是有关细菌借助菌毛在底物表面的蹭行运动及滑行运动的研究不是特别清楚，另外，无鞭毛降解菌是如何克服石油烃分子的高疏水性与之接触及无鞭毛菌在土壤环境中是否具有趋化性，目前还有待进一步深入研究。所以，加强细菌在底物表面的运动模式和趋化机制的研究是很有必要的。此外，近些年关于趋化的研究主要集中在趋化现象的表征上。多数受体蛋白对趋化物的感应机制不是十分清晰。因此，未来仍需深入研究微生物对趋化物的感应受体蛋白，以阐明其感应机制。
1.2 微生物对石油烃的转运过程石油烃大多通过被动运输、主动运输和内吞的方式进入细胞，部分小分子量的石油烃可以通过自由扩散的方式进入细胞。大部分石油烃的转运过程需要转运蛋白的参与，但是，转运蛋白的克隆及异源表达有一定的难度，而发现新的转运蛋白需要一定的时间[1]，因此，基于现有的报道，研究并阐明转运蛋白的序列、结构、底物和转运活性之间的一般性关系是很有必要的。
2 石油烃的生物降解过程微生物通过分解石油烃污染物将其同化为细胞生物量。目前报道的石油降解菌大多为野生型菌株，然而近些年随着基因工程、酶工程、合成生物学等技术的迅猛发展，研究者逐步开展对石油烃降解菌的工程改造等研究，期望获得生物修复效果更好的石油烃降解菌株[9-10]。
2.1 野生型菌株及其降解石油烃路径2.1.1 野生型菌株目前，有关从环境中分离石油烃降解菌，并利用微生物修复石油污染环境的工作已有很多报道。研究发现，200多种微生物有降解石油烃污染物的能力，包括细菌、真菌和藻类等[11]。表 1列举了部分降解烷烃的菌属。研究人员从原油污染现场筛选出了许多具有良好原油降解能力的天然单一菌株。例如，从土壤中分离出的芽孢杆菌Bacillus salamalaya 139SI在含2%和1%原油中培育42 d，其原油降解率分别为79%和88%[12]。从石油烃污染土壤中分离得到降解正十六烷和芘的红球菌Rhodococcus sp. T1，分别在以含2%十六烷和200 mg/L芘为碳源的培养基中培养5 d，其降解率分别为90.81%和42.79%[13]。
表 1 部分降解烷烃的菌属Table 1 Part of the alkane-degrading bacteria
| Bacteria | Carbon chain length | References | Fungi | Carbon chain length | References |
| Mycobacterium sp. | C7–C30 | [14] | Graphium sp. | Short chain | [15] |
| Rhodococcus sp. | C10–C19 | [16] | Talaromyces sp. | C6, C8 | [17] |
| Ochrobactrum sp. | C11–C29 | [18] | Aspergillus sp. | C7–C30 | [14] |
| Brevibacterium sp. | C12–C34 | [19] | Fusarium sp. | C8–C40 | [20] |
| Micrococcus sp. | C14–C33 | [21] | Penicillium sp. | C20, C22 | [22] |
| Pseudomonas sp. | C14–C35 | [23] | Purpureocillium sp. | C14 | [24] |
| Dietzia sp. | C16 | [25] | Neosartorya sp. | – | [26] |
| Acinetobacter sp. | Medium and short chains | [27] | Pleurotusostreatus sp. | – | [28] |
| Bacillus sp. | > C36 long chain | [29] | Trametesvillosus sp. | – | [30] |
| Alcaligenes sp. | – | [31] | Candida sp. | – | [32] |
| Gordonia sp. | – | [33] | Pichia sp. | – | [34] |
| Flavobacterium sp. | – | [35] | Yarrowia sp. | – | [36] |
| Note: “–” indicates that the length of the carbon chain of the alkane was not specified in the literature. | |||||
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但是，石油烃污染物组分复杂、降解途径冗长，使用单一菌株降解石油烃常面临缺乏广泛的基因修饰空间、底物范围窄以及降解效率较低等问题，因此，可以利用基因工程和代谢工程等手段，对野生型石油烃降解菌进行改造，或者构建石油烃降解的混菌体系，进一步提升其对石油烃污染环境的生物修复能力。
2.1.2 石油烃的降解路径目前，石油烃的部分微生物降解途径已经得到充分研究。
直链烷烃主要通过单末端氧化、双末端氧化、次末端氧化、ω-氧化和β-氧化等5种方式降解[37]。正十二烷在有氧条件下首先被烷烃单加氧酶氧化为初级醇，初级醇经醇脱氢酶的氧化生成醛，然后再经过醛脱氢酶生成酸，最后生成乙酰辅酶A (图 3A)[38]；支链烷烃结构稳定，一般通过末端氧化或β-氧化的方式降解。有研究人员通过分析由姥鲛烷代谢产生的中间体，提出了姥鲛烷的降解路径，并证实了姥鲛烷不仅可以通过单末端氧化和双末端氧化途径降解，还可以通过第3个碳原子上的亚末端氧化降解(图 3B)[39]。
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| 图 3 正十二烷(A)、姥鲛烷(B)、环己烷(C)、萘(D) 和菲(E) 的有氧降解[38-40, 61-62] Fig. 3 Aerobic degradation of n-dodecane (A), pristane (B), cyclohexane (C), naphthalene (D), phenanthrene (E)[38-40, 61-62]. |
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环烷烃的降解难度一般大于链烷烃，其降解需要多种不同的烷烃氧化酶协同参与。不动杆菌有氧降解环己烷的生化和遗传研究表明，前后共有两个操纵子编码的5种酶参与了降解，依次是单加氧酶、环己醇脱氢酶、环己酮单加氧酶、己内酯水解酶和醛脱氢酶(图 3C)[40]。环己烷在硫酸盐条件下厌氧降解时，依次发生延胡索酸盐加成、C骨架重排、脱羧、β-氧化等反应[41]。
芳香烃的降解已经得到了充分研究。在芳香烃的好氧降解中，菌株主要有假单胞菌属Pseudomonas sp.[42]、纤孔菌属Bjerkandera sp.[43]、芽孢杆菌属Bacillus sp.[44]等；酶主要有单加氧酶、双加氧酶(C23O[45]，NahAaAbAcAd[46]) 等；基因主要有单加氧酶基因(phe[47]) 和双加氧酶基因(nahAC[48]，nidA[49]，c12o[50]，nag，nar，phn，bph[51])；降解的机理是加氧酶将氧原子加到C-C键上形成C-O键，通过加氢、脱水等作用使C-O键断裂，从而使苯环裂解[52]；降解的途径是多环芳烃经双加氧酶作用生成二氢二醇化合物，在脱氢酶作用下生成二醇中间产物，随后通过内环断裂双加氧酶或外环断裂双加氧酶生成中间产物(如邻苯二酚)，最终进入三羧酸(TCA) 循环。多环芳烃经单加氧酶作用生成一种不稳定的芳香氧化物，经环氧化物酶催化生成反-二氢二醇化合物[42]。在芳香烃的厌氧降解中，菌株主要有假单胞菌属Pseudomonas sp.[53]、厚壁菌属Firmicute sp.[54]、梭菌属Clostridium sp.[55]等；酶主要有合酶(苄基琥珀酸合酶、萘甲基琥珀酸合酶)、羧化酶、脱氢酶[56]、还原酶(BcrCBAD/BzdNOPQ/BadDEFG, BamBCDEFGHI[57])；基因主要有合酶基因(bssA, nmsA[58]) 以及bamB、bamA、bcrA、bcrC、bzdN、ncr[56]等；厌氧降解的机理是多环芳烃与酶或其他物质相结合发生羧化反应、还原反应、羟基化及甲基化反应等使多环芳烃开环[52]；降解途径是厌氧微生物以硝酸盐、硫酸盐、铁、锰、二氧化碳等作为电子受体，将多环芳烃转化为小分子化合物，进一步转化为二氧化碳和甲烷[59]。多环芳烃通过甘氨酰基自由基酶所产生的芳香琥珀酸，进一步通过甲基化反应、羧基化反应、羟化反应，最终进行β-氧化实现多环芳烃的降解[60]。萘在有氧条件下，经加氧酶的作用生成中心中间产物(邻苯二酚、原儿茶酸、龙胆酸盐和尿黑酸等)，而后经开环进入TCA循环(图 3D)[61]；菲在有氧条件下经过双加氧酶、脱氢酶、异构酶、水合-醛缩酶等一系列酶的作用下生成1-羟基-2-萘甲酸，而后1-羟基-2-萘甲酸在双加氧酶、水合-醛缩酶、脱氢酶、脱羧酶等作用下生成原儿茶酸，最终进入TCA循环(图 3E)[62]。菲在无氧条件下，经合酶、羟酶、脱氢酶等作用生成中心中间产物(如苯甲酰辅酶A及间苯二酚、间苯三酚、羟氢鲲等其衍生物)，随后经还原酶生成乙酰辅酶A[63]。表 2列举了芳香烃的好氧和厌氧降解在降解菌、酶、基因、降解机理和降解途径等方面的对比。
表 2 芳香烃的好氧和厌氧降解比较Table 2 Comparison of aerobic and anaerobic degradation of aromatic hydrocarbons
| Aerobic degradation | Anaerobic degradation | |
| Typical degrading strains | Pseudomonas sp.[42] Bjerkandera sp.[43] Bacillus sp.[44] | Pseudomonas sp.[53] Firmicute sp.[54] Clostridium sp.[55] |
| Degrading enzymes | Monooxygenase, dioxygenase (C23O[45], NahAaAbAcAd[46]) | Synthase (benzylsuccinate synthase, naphthylmethylsuccinate synthase), carboxylase, dehydrogenase, reductase (BcrCBAD/BzdNOPQ/BadDEFG, BamBCDEFGHI[57]) |
| Degrading genes | Monooxygenase genes (phe[47]), Dioxygenase genes (nahAC[48], nidA[49], c12o[50], nag, nar, phn, bph[51]) | Synthase genes (bssA, nmsA[58]) bamB, bamA, bcrA, bcrC, bzdN, ncr[56] |
| Degrading mechanisms | Oxygenase adds oxygen atoms to the C-C bond to form the C-O bond, and breaks the C-O bond through hydrogenation, dehydration and other actions, thus cracking the benzene ring | The combination of PAHs with enzymes or other substances leads to carboxylation, reduction, hydroxylation and methylation reactions, in order to crack the rings of PAHs |
| Degrading pathways | PAHs produce dihydrodiol compounds by dioxygenase and diol intermediates by dehydrogenase. Then intermediate products (such as catechol) are produced by inner ring or outer ring breaking dioxygenase, and then enter the TCA cycle PAHs produce an unstable aromatic oxide by monooxygenase. Then, epoxides catalyze it to produce anti-dihydrodiol compounds[42] | Using nitrates, sulfates, iron, manganese, and carbon dioxide as electron receptors, anaerobic microorganisms convert PAHs into small molecular compounds and further convert them into carbon dioxide and methane PAHs produce aromatic succinic acid catalyzed by glycyrrhizin free radical enzymes, then through methylation reaction, carboxylation reaction, hydroxylation reaction and finally β-oxidation[60] |
表选项
2.2 工程改造菌株从自然界中分离出的菌株降解效率较低、自然降解过程较慢，实际应用中需要提高修复效果。基因工程、酶工程、蛋白质工程等方式是比较有效的强化菌株的手段[64]。将承载多种降解基因的质粒导入到同一菌株中，可获得具有多种底物降解潜力的重组基因工程菌。虽然大多数石油烃降解菌的遗传背景不清楚，有关基因工程等手段改造石油烃降解菌在生物修复领域内的研究还不是很深入，但此项技术在该领域有着巨大的发展潜力。
鉴于石油烃是各种烃类的混合物，利用基因工程技术构建能够降解各种石油烃类的工程菌是生物修复石油烃污染环境的发展方向。近几年来，国内外****对高效降解石油烃的基因工程菌已展开了部分研究。微生物对某些石油烃成分的降解是通过质粒控制的，因此，可以通过引入具有降解不同成分能力的质粒在单一细胞中来构建超级细菌。例如，从恶臭假单胞菌P. putida BNF1菌株中克隆得到xylE基因，将其插入到不动杆菌Acinetobacter sp. BS3的染色体中并表达。构建得到的工程菌株和原始菌株相比，具有更广泛的底物特异性、具有更高的降解多种正构烷烃和芳香烃的能力[65]。Luo等[66]将假单胞菌P. putida GPo1的烷烃羟化酶基因转入到大肠杆菌中，显著提高了石油降解菌与大肠杆菌混合菌群的降解率。研究人员通过对筛选出的拜氏不动杆菌Acinetobacter beijerinckii TY22中的烷烃羟化酶基因进行PCR扩增、构建重组质粒、进行基因功能互补试验，最终在大肠杆菌中成功表达了该基因[67]。除此之外，还有研究者将荧光素酶基因lux插入到具有降解石油烃能力的鲍曼不动杆菌Acinetobacter baumannii S30的染色体上，重组得到鲍曼不动杆菌A. baumannii S30pJES，从而达到在生物修复位点监测其存活情况的目的[10]。
近几十年来，大量的研究致力于探索有效的方法对多环芳烃污染进行生物修复。从多环芳烃污染的水和土壤中分离出具有明显降解多环芳烃能力的天然菌株，鞘脂菌属和鞘脂醇单胞菌属由于潜在的生物降解能力和多样化的生态适应性而得到广泛研究。尽管已知它们能够降解一系列芳香族化合物，但其降解代谢物、降解相关基因以及调节因素和机制等仍需要进一步阐明。为了深入了解微生物的修复能力，Zhao等[68]最近对26株鞘脂菌属和鞘脂醇单胞菌属的细菌进行了全基因组测序。在6个多环芳烃的菌株中鉴定出bph和xyl基因簇，根据26个基因组中大量基因编码加氧酶的结果进行预测，表明这些菌株具有明显的生物修复潜力。通过融合芘降解菌假单胞菌Pseudomonas sp. GP3A和菲降解菌鞘氨醇单胞菌Sphingomonas sp. GY2B获得了能够降解多环芳烃的融合体F14[69]。
另外，把代谢工程的方法运用到石油烃的降解上，开发石油烃降解菌的分子操作工具，对石油烃降解菌进行合理的改造和调控。过表达代谢途径的相关基因作为一种静态调控的主要策略在石油烃降解菌产表面活性剂方面的应用相当广泛。有研究表明，过表达脂肪酸合成途径中相关基因均能促进表面活性素(一种脂肽类生物表面活性剂) 合成，说明过表达相关基因是提高菌株表面活性素合成能力的有效措施[70]。Wu等[71]在枯草芽孢杆菌Bacillus subtilis 168中通过整合表面活性素合成激活因子、敲除竞争途径、强化脂肪酸前体供应等多个策略协同改造，从而使表面活性素产量显著提高。
随着合成生物技术的快速发展，利用模式菌株作为底盘细胞，开发标准化的石油降解底盘细胞，在底盘细胞中构建石油降解功能模块，有望实现目标化合物的降解。在菌株的调控方面，除了采用基因敲除和过表达等静态调控策略外，还可以构建调控元件、设计基因线路等精确调节物质流及能量流平衡，从而避免因代谢工程改造导致的细胞代谢流与能量流失衡、生长阻滞和毒性中间体积累等问题。笔者课题组近几年在合成生物学领域取得了重要的研究进展[72-73]，从不同合成型菌株出发，大幅提升了细胞的耐受性，从而获得了优良的底盘细胞[74]；借助SCRaMbLE系统设计构建了缺失YEL013W的合成型单倍体菌株，该菌株生产类胡萝卜素的产量增加[75]；以含有环形染色体的酿酒酵母菌株为模型，利用SCRaMbLE系统诱导环形染色体的基因组重排，迅速提升了紫色杆菌素前体的产量[76]。所以，基因组再造与重排技术也可以为石油烃降解中如何快速获得具有优良性状的底盘细胞提供新策略，进而实现菌株的快速进化、代谢通路的快速优化等。
2.3 构建降解石油烃的混菌体系从石油烃污染地点分离石油烃降解菌群已经得到了充分研究。例如，Wang等[77]分离培育出的微生物群落在30 d内迅速降解原油；另有研究者通过逐级驯化得到一组对稀油和稠油均具有降解能力的混菌M3，该混菌M3显著促进了土壤中原油的降解[78]。
除了从环境中分离出土著的石油烃降解菌群之外，更多的研究侧重利用土著菌构建复配的混合菌群。复配的两菌体系在明显提升石油烃降解效率的同时往往具有协同作用，利用多种菌株复配出的混菌体系也能达到良好的效果。例如，吴霜等复配出的三菌体系通过菌种间的协同作用显著提高菲的降解效率[79]；两株促生长的细菌通过促进真菌菌丝的生长，从而提高了该三菌体系对多环芳烃的降解[80]。有研究人员复配出的四菌体系在降解萘和蒽的同时，还能生产表面活性剂[81]；由分离出的6株石油烃降解菌组成的混合菌群对C8–C35的降解率显著高于单个降解菌的降解率[82]；利用8株霉菌、3株酵母菌和4株细菌分别采取细菌混合培养、真菌混合培养、细菌和真菌混合培养等不同的组合方式进行石油烃降解实验[83]。以上结果表明，具备协同作用的微生物菌群的修复效果一般会显著高于单一菌株。可见，开发出具有强大降解石油烃潜力的微生物混菌体系至关重要。
但是，现有的石油烃降解菌群的研究大多集中在从石油环境分离出的土著菌群或者利用土著菌复配的混合菌群，存在菌株间分工不明确、代谢不平衡、竞争与协作共存和相互作用关系错综复杂等问题。近年来，利用合成生物学等方法对各个菌株进行有目的的设计改造，从而构建人工混菌体系逐渐成为研究热点。人工构建的混菌体系可以减轻单个底盘细胞的代谢负担、降低中间代谢物的过度积累和毒害、避免功能间的交叉影响以及对环境波动具有更强的适应性和鲁棒性等，所以在实现复杂的生物功能方面具有明显优势[84]。在人工混菌体系的研究中，依据劳动分工原则构建人工混菌体系，可将复杂的任务合理分配给不同的菌株，在一定程度上避免单菌中细胞代谢负荷重等问题，在提高效率等方面具有显著优势。例如，依照“劳动分工合作”原则构建出了用于产电的互利共生人工三菌体系，并通过调控电子载体的合成、优化碳源等，使该体系仅利用0.28 g葡萄糖就能稳定产电15 d以上[85]。当存在竞争关系时，可以通过重构混菌体系中的菌株关系从而解除竞争抑制，达到互利共生的关系，菌株之间共享或交换营养物质，并分别从中获益。依据微生物间的互作关系，采用模块化构建的方法，有针对性地设计和重构各个功能菌种，通过构建互利共生的人工混菌系统，使菌株促进彼此的生长，可进一步提升人工混菌体系的高效性和稳定性。例如，依据互利共生原理，本课题组成功设计重构了维生素C两菌一步发酵体系和三菌一步发酵体系，实现了菌株之间由偏利共生关系和竞争关系到互利共生关系的转变[86-87]。
人工混菌体系在环境治理方面的应用潜力巨大，通过构建两株假单胞菌混菌体系，可提高对石油硫化物的脱硫作用[88]。人工构建的大肠杆菌-大肠杆菌混菌体系，可以有效降解杀虫剂[89]。因此，可以带有明确目的地构建互利共生的石油烃降解人工混菌系统，使菌株促进彼此的生长，从而提高该体系的石油烃降解能力。例如，有研究者设计构建了由大肠杆菌Escherichia coli HY1和铜绿假单胞菌P. aeruginosa PH2组成的分工明确的混菌体系用于菲的降解，在大肠杆菌E. coli HY1中构建了两个末端双加氧酶模块和一个电子转移链，在铜绿假单胞菌P. aeruginosa PH2中构建了一个邻苯二酚1, 2-双加氧酶模块，该混菌体系首先将菲氧化为9, 10-二羟基菲和1, 2-二羟基菲，然后通过邻苯二酚将这些中间体代谢为二氧化碳和水，在此过程中，大肠杆菌E. coli HY1负责菲的初始氧化过程，铜绿假单胞菌P. aeruginosa PH2负责环的裂解[90]，由此该混菌体系实现了“劳动分工合作”。
因此，在构建石油烃降解菌的混菌体系时，也可以借助上述思想，将真菌、细菌等设计构建成人工混菌体系，可使部分菌株分工，如不同菌株主要降解不同种类的石油烃，部分菌株主要产表面活性剂，部分菌株主要实现物质转运和电子转运等，使混菌体系可以实现对不同类型石油烃的有效降解功能。在混菌体系的构建过程中，各菌株之间的适配是实现其稳定发挥功能的必要因素之一，其实现过程需要在物质代谢和能量代谢水平上，反复进行菌株与菌株之间的适配，这也是合成生物学领域要解决的关键问题之一。
3 展望石油烃成分复杂，微生物对其降解路径冗长，从环境中分离得到的石油烃降解菌降解效率较低、自然降解过程较慢，分离出的混合菌群存在竞争抑制、相互作用关系错综复杂等问题。对石油烃降解菌代谢路径的部分基因进行敲除、过表达等改造，不仅可以强化代谢路径，而且可以使石油烃降解菌更容易适应实际油藏环境；对菌株的石油烃降解路径进行合理改造和调控，通过构建调控元件、设计基因线路等手段精确调节物质流及能量流平衡，将显著提升降解菌的降解能力；将真菌、细菌等设计构建成石油烃降解的人工混菌体系，可使部分菌株分工实现对不同类型石油烃的降解功能。借助代谢工程和合成生物学的研究思想，对分离出的石油烃降解菌进行基因工程改造、构建人工混菌体系等可望成为石油烃复杂成分降解的有效策略。
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