【作者】 郭强；

【导师】 邓利；

【作者基本信息】 北京化工大学 ， 生物工程， 2022， 博士

【摘要】 微生物发酵是绿色生物制造的重要技术手段,其核心在于高效细胞工厂的构建。大肠杆菌具有遗传背景清晰、技术操作简单、生长繁殖快速、大规模发酵方便等优点,已成为细胞工厂的优秀底盘宿主。利用代谢工程理性设计细胞代谢途径可有效提高糖等底物的转化效率,而糖的跨膜转运是其进入代谢的第一步。大肠杆菌对糖的运输具有专一性,它依赖于细胞质膜上的特异性转运蛋白,并以主动运输或协助扩散的方式完成。这一特点不仅会导致底物范围狭窄,还会引发“饱和现象”、抑制物敏感、碳分解代谢物阻遏（CCR）等问题。最理想的解决方案是在不过度干扰细胞生命活动的前提下,开发高效的糖扩散通道,促使糖分子利用细胞膜两侧的浓度差,通过简单扩散方式进入细胞质。大肠杆菌的SecY蛋白转位通道定位于细胞质膜,其突变复合体SecY（ΔP）EG（删除栓塞结构域）与细胞外膜致孔蛋白（SCVE）共同表达可实现单糖分子简单扩散跨膜。尽管如此,SecY（ΔP）通道的单糖转运效率及优势尚不明确,且其在细胞工厂中的应用未曾被开发。此外,目前SecY（ΔP）通道运输乳糖等二糖的能力仍较差。基于上述问题,本论文首先对SecY（ΔP）通道转运单糖的优势进行评价。其次,将通道应用于木糖醇合成途径,实现碳代谢流自动控制。同时,借助SecY（ΔP）通道建立了D-阿洛酮糖的发酵制备法。最后,定点突变通道的氨基酸孔环,有效改善了乳糖转运效果。具体内容和结果如下:1.SecY（ΔP）通道转运单糖的优势评价。利用C¹⁴同位素示踪技术证实SecY（ΔP）通道可高效转运单糖。使用SecY（ΔP）通道替代葡萄糖PTS的重组菌株在100 g/L葡萄糖上的比生长速率为0.228 h^-1,比野生型高80%,打破了转运“饱和现象”。当存在葡萄糖竞争性抑制剂时,重组菌株生长不受影响。在混糖（木糖和葡萄糖）发酵实验中,重组菌株实现了糖的同步利用,且比生长速率从0.174 h^-1提高到0.309 h^-1。由上可知,SecY（ΔP）通道转运单糖比特异性转运蛋白更具优势,为其后续的应用研究提供了理论基础。2.借助SecY（ΔP）通道实现木糖醇高效合成过程的碳代谢流自动控制。木糖发酵实验表明,利用SecY（ΔP）通道能彻底解除木糖醇-磷酸对细胞转运木糖的抑制问题。引入木糖醇合成途径后,在SecY（ΔP）通道与CCR效应的协同作用下进行混糖（木糖和葡萄糖）发酵实验。当葡萄糖存在时,木糖瞬时消耗率≈0.81 m M·h^-1·OD^-1,木糖醇瞬时得率≈0.72mol/mol。当葡萄糖耗尽后,木糖瞬时消耗率≈1.51 m M·h^-1·OD^-1,木糖醇瞬时得率≈0.25 mol/mol,表明细胞可自动调节木糖代谢通量。新型细胞工厂在木糖醇合成过程中,不仅实现了葡萄糖和木糖的同步转运,而且能够根据葡萄糖的浓度变化自动调整木糖的代谢流分配,有效提高了产物木糖醇的得率。3.借助SecY（ΔP）通道实现D-阿洛酮糖合成的底物非磷酸化转运。利用SecY（ΔP）通道和D-阿洛酮糖3-差向异构酶（DPEase）成功构建以果糖为底物合成D-阿洛酮糖的细胞工厂,得率≈0.05 g/g。依次敲除底物磷酸化途径和副产物合成途径,D-阿洛酮糖得率提高到≈0.59 g/g。进一步调控糖酵解途径的碳代谢通量,D-阿洛酮糖得率最终可达到≈0.95g/g。筛选发酵培养基后,使用补料分批发酵的方式制备D-阿洛酮糖,最终产量≈23.3 g/L,对数生长期的时空产率≈1.03 g/（L·h）。4.SecY（ΔP）通道的氨基酸孔环突变及其乳糖转运功能探索。绿色荧光蛋白（GFP）表达实验证明SecY（ΔP）通道可微量转运乳糖。为提高通道的乳糖扩散效率,利用定点非随机突变将其氨基酸孔环的6个异亮氨酸（Ile）全部突变为缬氨酸（Val）,突变菌株E.coli（ΔLac Y,SecY[ΔP,Val],SCVE）利用乳糖为唯一碳源的生长周期明显缩短。在此基础上,通过定点随机突变筛选,得到了突变菌株E.coli 2RM53,其在对数生长期的比生长速率进一步提高了13.1%。结果说明对SecY（ΔP）孔环氨基酸的适当调整,可有效提高其乳糖转运能力,为利用大肠杆菌的SecY（ΔP）通道转运其它二糖分子奠定了基础。综上所述,基于SecY（ΔP）通道建立的简单扩散跨膜系统能够有效克服大肠杆菌特异性糖转运系统的缺陷,并被成功应用于合成木糖醇和D-阿洛酮糖的细胞工厂。氨基酸孔环突变后的SecY（ΔP）通道扩散乳糖的效率显著提高,后续有望作为大肠杆菌的非特异性二糖转运通道。更多还原

【Abstract】 Microbial fermentation plays an important role in green bio-manufacturing,in which the construction of efficient cell factories is known as the major point.Escherichia coli has become a widely-used bacterial host due to its clear genetic background,simple manupilation,rapid growth,and easy large-scale fermentation.Rational design of intracellular pathways through metabolic engineering is one of the ways to improve the performance of cell factories,and sugar transport is a prerequisite for its further metabolism inside cells.The uptake of sugars by E.coli relies on a variety of specific transporters on its cytoplasmic membrane,which function in either an active transport or facilitated diffusion manner,leading to narrow sugar spectrum,transport saturation phenomenon,competitive/noncompetitive inhibition,and carbon catabolite repression（CCR）.The ideal solution is to develop an efficient simple diffusion channel without excessively interfering with cell growth,allowing sugar molecules to enter the cytoplasm under the concentration difference between the two sides of the membrane.The SecY protein-translocation channel（SecYEG）of E.coli is located on the cytoplasmic membrane.We have previously reported that co-expression of the SecY（ΔP）channel（a mutant without plug domain）and the outer membrane porin（SCVE）could form a passageway for monosaccharides to freely diffuse into the cytoplasm.However,the advantages of the SecY（ΔP）channel for transporting monosaccharides when compared with the specific transporters are still unclear,and its applications have not yet been fully explored.Moreover,the transport ability of the SecY（ΔP）channel for disaccharides,such as lactose,is quite weak.In this work,we evaluated the advantages of SecY（ΔP）channel for uptake of monosaccharides.Then,the SecY（ΔP）channel was applied to automatically control the carbon flux in cells for efficient synthesis of xylitol.After that,fermentative production of D-allulose was accomplished with a SecY（ΔP）channel-engineered cell factory.Finally,site-directed mutagenesis of the pore ring of the SecY（ΔP）channel was investigated to improve the uptake performance for lactose.The detailed contents are as follows.1.Evaluation of the advantages of SecY（ΔP）channel for transport of monosaccharides.We constructed the SecY（ΔP）channel in E.coli,and then demonstrated its ability to transport monosaccharides using C¹⁴-labeled glucose.The engineered E.coli with the SecY（ΔP）channel rather than the glucose PTS could maintain a specific growth rate of 0.228 h^-1 on 100 g/L glucose,which was 80%higher than that of the wild type E.coli,suggesting that the glucose transport saturation has been broken.The growth of mutant cells was not affected when competitive inhibitors of glucose were present in the medium.In fermentation of xylose-glucose mixtures,the engineered E.coli achieved simultaneous utilization of sugars,and the specific growth rate was increased from 0.174 h^-1 to 0.309 h^-1.Therefore,it can be seen that the SecY（ΔP）channel has obvious superiority in uptake of monosaccharides,which provides an important theoretical basis for subsequent applications.2.Intelligent self-control of carbon metabolic flux for xylitol synthesis by use of SecY（ΔP）channel.Fermentation experiments of xylose showed that xylitol-phosphate could no longer inhibit the uptake of xylose by cells with the help of SecY（ΔP）channel.After introduing the xylitol synthesis pathway,we used mixed sugar（xylose and glucose）for fermentation under the synergistic effect of SecY（ΔP）channel and CCR.In the presence of glucose,the instantaneous consumption rate of xylose was≈0.81 m M·h^-1·OD^-1,and the instantaneous yield of xylitol was≈0.72 mol/mol.After glucose was exhausted,the instantaneous consumption rate of xylose was≈1.51 m M·h^-1·OD^-1,and the instantaneous yield of xylitol was≈0.25 mol/mol.These results suggest that cells were able to automatically regulate the metabolic flux of xylose.The novel cell factory for xylitol synthesis not only realized the simultaneous transport of glucose and xylose,but also could automatically adjust the distribution of xylose flux according to the changes of glucose level,thus ensuring a high product yield.3.Transport of substrate without phosphorylation by the SecY（ΔP）channel for D-allulose synthesis.A synthetic pathway for D-allulose was constructed using the SecY（ΔP）channel and D-psicose 3-epimerase（DPEase）,resulting in a yield of≈0.05 g/g through fermentation.Then,knockout of PTS,fructokinase（Mak）and by-product pathways was carried out,increasing the yield of D-allulose to≈0.59 g/g.Further regulation of the carbon metabolic flux of the Embden-Meyerhof-Parnas（EMP）pathway got a final D-allulose yield of≈0.95 g/g.After medium optimization,the D-allulose titer reached≈23.3 g/L via fed-batch fermentation,in which the productivity throughout the logarithmic growth phase was≈1.03 g/（L·h）.4.Tageted mutation of the pore ring of SecY（ΔP）channel and its functionality in lactose transport.The experiment of green fluorescent protein（GFP）expression demonstrated that the SecY（ΔP）channel allowed slow passage of lactose.In order to increase the diffusion rate of lactose,mutation of its pore ring domain was carried out.First,a site-directed non-random mutagenesis method was developed.When all of the six isoleucines（Ile）of the pore ring were substituted with valine（Val）,the growth of the mutant E.coli（ΔLac Y,SecY[ΔP,Val],SCVE）on lactose was obviously accelerated.Next,we further performed site-directed random mutagenesis and obtained a mutant strain named E.coli 2RM53,whose specific growth rate during the logarithmic growth phase was increased by 13.1%when compared with E.coli（ΔLac Y,SecY[ΔP,Val],SCVE）.The results indicate that appreopriate adjustment of the amino acids of the SecY（ΔP）pore ring would effectively improve the lactose transport performance of the channel,thus laying a foundation for the use of the SecY（ΔP）channel of E.coli to take up other disaccharides.In summary,a free diffusion system established in E.coli using the SecY（ΔP）channel was able to overcome the defects of the native sugar transport systems,and it was successfully applied to the cell factories for production of xylitol and D-allulose.Mutation of the pore ring of the SecY（ΔP）channel could obviously improve its lactose transport efficiency,indicating that the SecY（ΔP）channel might be used in E.coli as a non-specific transporter for uptake of disaccharides.更多还原