基于工程化盐单胞菌的下一代工业生物技术

doi:10.12211/2096-8280.2020-042

摘要/Abstract

摘要：

我国是工业生物技术大国，拥有世界上最大的发酵产业，但传统发酵需要灭菌操作，发酵过程高耗能、耗淡水且不能连续，导致生产成本偏高，无法与化学工业竞争。因此，急需开发下一代工业生物技术（NGIB）来克服这些缺点。NGIB利用盐单胞菌等极端微生物作为底盘细胞，具有发酵不需灭菌、节能节水、设备投资少、产物终浓度高、分离过程简单等优点。本文围绕下一代工业生物技术的发展过程，系统介绍了近年来在技术优势强化，生物元件和工具开发如Porin启动子系统、CRISPRi系统、CRISPR-Cas9系统等，新产物合成如聚（3-羟基丁酸-co-3-羟基戊酸）（PHBV）、聚（3-羟基丁酸-co-4-羟基丁酸）（P3HB4HB）、表面活性剂蛋白等，以及PHA分离过程优化、发酵工艺放大、废水循环利用等方面取得的最新进展。随着合成生物学发展和应用，基于工程化盐单胞菌的下一代工业生物技术体系正在不断完善，优势也愈加明显。下一代工业生物技术将为大幅提升绿色生物制造的竞争力提供强力支撑。

关键词: 下一代工业生物技术（NGIB）, 盐单胞菌, 生物制造, 发酵工业, 聚3-羟基丁酸（PHB）, 生物塑料, 聚羟基脂肪酸酯（PHA）

Abstract:

China is a country with the largest industrial biotechnology capacity especially fermentation. However, it requires complicated sterilization procedures,high consumption of fresh water and energy, and expensive wastewater treatment processes. Therefore, it is urgent to develop the "Next Generation Industrial Biotechnology (NGIB)". The NGIB based on Halomonas spp. has many advantages, including: (1) energy-saving: no need for sterilization at high temperature and high pressure; (2) water-saving: use seawater instead of fresh water, and water can be recycled; (3) time-saving: production process can be continuous; (4) less investment in equipment: no need to use stainless steel fermentation system, instead, plastic, ceramics or even cement can be used as bioreactors; (5) high concentration of final product, bacteria can produce products at a high concentration; (6) simplification of separation process: increase bacterial volume or surface charge, which is conducive to gravity or self-flocculation precipitation. Based on the newly developed ‘Next Generation Industrial Biotechnology', this paper systematically introduces the latest progress in recent years in the fields such as strengthening technical advantages, development of biobricks and control parts including the porin promoter library, CRISPRi, CRISPR/Cas9 gene editing system, production of new products including poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV), Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)(P3HB4HB) and Bio-surfactant Protein PhaP, optimization of separation process, scale up of fermentation processes, and recycling of wastewater. With the application of synthetic biology, the efficiency of NGIB has been continuously improved, and its advantages are obvious. Nevertheless, NGIB also faces some technical challenges, particularly treatment of salt containing wastewater. NGIB will further improve the recycling strategy of high salt wastewater, develop control modules under high cell density fermentation conditions, strengthen the design of low-cost substrate utilization ways, expand the scale and application fields, and achieve the mass production of various chemicals. The continuous improvement of NGIB will provide strong support for the competitiveness of green bio-manufacturing.

Key words: next generation industrial biotechnology, Halomonas spp., biological manufacturing, fermentation industry, PHB, bioplastics, PHA

中图分类号:

Q 819

陈江楠, 陈潇宁, 刘心怡, 万薇, 章义鑫, 张自豪, 郑逸飞, 郑陶然, 王宣, 王子瑜, 闫煦, 张旭, 吴赴清, 陈国强. 基于工程化盐单胞菌的下一代工业生物技术[J]. 合成生物学, 2020, 1(5): 516-527.

CHEN Jiangnan, CHEN Xiaoning, LIU Xinyi, WAN Wei, ZHANG Yixin, ZHANG Zihao, ZHENG Yifei, ZHENG Taoran, WANG Xuan, WANG Ziyu, YAN Xu, ZHANG Xu, WU Fuqing, CHEN Guoqiang. Engineering Halomonas spp. for next generation industrial biotechnology （NGIB）[J]. Synthetic Biology Journal, 2020, 1(5): 516-527.

图/表 4

图1 基于盐单胞菌的下一代工业生物技术的流程[9]

Fig. 1 Process of next generation industrial biotechnology based on Halomonas spp.[9]

图2 盐单胞菌H. bluephagenesis中已开发的下一代分子生物技术工具（a）、方法（b）和合成的多种产物（c）[5]1—Porin启动子表达系统；2—T7-like诱导表达系统；3—CRISPRi基因编辑系统；4—CRISPR/Cas9基因编辑系统；5—提升氧气利用率；6—形态工程改造；7—高密度生长；8—大规模发酵；9—PHA产品类型（PHB、PHBV和P3HB4HB）；10—其他产品类型（PhaP，PhaR，ALA和ectoine）gRNA—向导RNA;PHBV—聚3羟基丁酸-3羟基戊酸酯;RBS—核糖体结合位点;sgRNA—单链向导RNA;VHb—透明颤菌血红蛋白

Fig. 2 Engineering halophilic Halomonas spp. for NGIB tools (a)， methods (b) and production of multiple products (c)1—Porin promoter expression system; 2—T7‐like expression system; 3—CRISPRi engineering system; 4—CRISPR/Cas9 engineering system; 5—increasing oxygen availability; 6—morphology engineering; 7—high‐cell‐density growth; 8—large‐scale fermentation; 9—PHA product types（PHB, PHBV, and P3HB4HB）; 10—other products （PhaP, PhaR, ALA, and ectoine）gRNA—guide RNA; PHBV—poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate); RBS—ribosome binding site; sgRNA—single‐guide RNA; VHb—Vitreoscilla hemoglobin

图3 利用盐单胞菌成功合成多种不同PHA材料[73]（a)使用T7-like诱导表达系统高产PHB；（b)利用非相关碳源在H. bluephagenesis TD08(prpC基因敲除)菌株生产PHBV；（c)利用γ‐丁内酯和葡萄糖在H. bluephagenesis TD40中生产P3HB4HB；（d)在H. bluephagenesis TD(phaC敲除)菌株中生产PhaP蛋白；（e)在H. bluephagenesis TD(phaC敲除)菌株中生产PhaR蛋白；（f)在加大或者加长的H. campaniensis LS21中(mreB基因敲除或者ftsZ基因敲除)生产PHB；（g)第一个5000L不灭菌开放连续塑料发酵罐，Halomonas spp.在发酵罐中生长；（h)在H. bluephagenesis TD LTT22中生产ALA基因：phaC—编码PHA合酶;phaA—编码β‐酮硫解酶;phaB—编码NADPH依赖乙酰乙酰辅酶A还原酶;prpC—编码2-柠檬酸甲酯合酶;orfZ—编码2-柠檬酸甲酯合酶；mreB—编码细胞骨架蛋白;ftsZ—编码细胞分裂蛋白;△ftsZ::ftsZ‐gfp—gfp基因插入ftsZ基因的C端，使后者丧失功能;hem1—编码ALA合酶

Fig. 3 Engineering halophilic Halomonas spp. for production of multiple PHA products(a) Production of PHB using T7‐like expression system in H. bluephagenesis TD‐HIGH；（b) Production of PHBV in H. bluephagenesis TD08 (without prpC) in the absence of propionic acid；（c) Production of P34HB in H. bluephagenesis TD40 using γ‐butyrolactone and glucose as carbon sources；（d) Production of PhaP in H. bluephagenesis TD (without phaC)；（e) Production of PhaR by H. bluephagenesis TD (without phaC)；（f) Production of PHB by enlarged or elongated H. campaniensis LS21 without mreB and deficient ftsZ, respectively；（g) The first 5000L plastic fermentor used to grow Halomonas spp. under open unsterile and continuous conditions；（h) Production of ALA in H. bluephagenesis TD LTT22Genes: phaC—encoding PHA synthase; phaA—encoding β‐ketothiolase; phaB—encoding NADPH dependent acetoacetyl‐CoA reductase; prpC—encoding 2‐methylcitrate synthase; orfZ—encoding 2‐methylcitrate synthase; mreB—encoding cytoskeleton protein; ftsZ—encoding cell division protein; △ftsZ::ftsZ‐gfp—a gfp gene was inserted into the C‐terminal of ftsZ to destroy its function; hem1—encoding 5‐amino‐levulinic acid synthase

表1 下一代工业生物技术与当前工业生物技术对比[9]

Tab. 1 Next generation industrial biotechnology (NGIB) compared with current industrial biotechnology

传统工业生物技术	下一代工业生物技术	实现途径
大量消耗淡水	对淡水依赖较低	海水或者循环用水
高能耗	低能耗	无灭菌开放式发酵、提高氧气利用率
易染菌	难染菌	筛选极端微生物
高成本投资	低成本投资	低成本设备
微生物生长条件苛刻	微生物生长条件灵活	筛选极端微生物
分批发酵	连续发酵	选择不易染菌的底盘菌
细胞不易分离	分离难度小	通过形态学工程改变细胞尺寸
发酵周期长	发酵周期短	利用合成生物学加快细胞生长
一个菌种对应一种产品	多个产品的平台菌	在平台菌中构建多条代谢途径
产物在胞内或者培养基中	实现胞内和胞外产品共产	工程化获得多种产品的共产
单一碳源培养生长	混合碳源或者混合废料生长	筛选或构建消耗混合碳源的菌种
自动化程度低	自动化控制智能化	菌种适应多种生长环境
底物有效转化率低	底物有效转化率高	敲除或者减弱代谢旁路
获得胞内产物难度大	减弱细胞壁	工程化细胞壁合成机制
胞外产物产量低	胞外产物产量高	减弱外膜结构

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