红色红球菌是丙烯酰胺工业生产的重要菌种，具有高产酶效率和高有机溶剂耐受性的特点，充分匹配工业生物催化过程对细胞催化剂的要求，具备发展为普适性全细胞催化宿主的潜力。然而，红色红球菌基因编辑工具匮乏，严重限制其工程化改造进程。本论文的目标是开发高效的红球菌基因编辑工具，面向产业化应用重组构建高性能的丙烯酰胺生产菌株，并拓展改造红色红球菌为普适性细胞宿主。首先在红色红球菌中引入cas9基因和sgRNA，建立基于CRISPR/Cas9的基因编辑方法，但发现其基因编辑过程受红球菌低转化效率和低重组效率的限制。通过规避限制-修饰系统，将转化效率提升了89倍；引入噬菌体重组酶Che9c60和Che9c61，显著提高了同源重组效率；最终在红色红球菌中建立了基于CRISPR/Cas9的dsDNA和ssDNA重组系统，成功实现基因敲除、插入和突变等改造，编辑效率最高达到75%。经过表达元件改进和新型重组酶RrRecT的挖掘与辅助重组，基于ssDNA的CRISPR/Cas9系统成功拓展应用至浑浊红球菌。基于sacB的同源重组双交换方法具有简单快捷的特点，但原方法在红色红球菌中存在单交换效率低、假阳性率高和蔗糖反向筛选效果差等问题，无法直接应用。本论文发现了自杀质粒抗性基因的决定性影响，通过抗性基因替换显著降低了假阳性率；强化sacB基因的表达，实现了高效的蔗糖反向筛选；分析并规避红球菌限制-修饰系统，提高了单交换效率。改进的sacB方法成功实现了红色红球菌的高效基因编辑（包括46.5 kb大片段敲除），单基因敲除周期从21天缩短至7天。利用上述基因编辑新方法，重组构建高性能丙烯酰胺生产菌株。敲除关键酰胺酶基因，彻底阻断副产物丙烯酸的合成；原位置换突变型腈水合酶基因，提高催化稳定性；敲除调控蛋白基因，强化腈水合酶表达，使酶活提高40%。成功构建了4株高性能的重组红球菌，应用于丙烯酰胺的工业化生产，节能减耗及高产效果显著。进一步以异源环氧化物水解酶、腈水解酶和转氨酶为对象，开发红色红球菌普适宿主，构建外源酶高效表达的高性能全细胞催化剂。以环氧化物水解酶为例，对比重组大肠杆菌，重组红球菌全细胞催化剂的环氧化物水解酶表达量占总蛋白的30%，且具有更高的热稳定性、pH稳定性、底物和产物耐受性。进一步基于腈水解酶和转氨酶的克隆表达与催化性能评价，建立了红色红球菌全细胞催化剂普适化开发的优选重组模式，证实了新方法的普适性和优越性。

Rhodococcus ruber is an important strain for bioproduction of acrylamide, and possesses high protein expression efficiency and high organic solvent tolerance. These characteristics make R. ruber an appealing whole-cell biocatalyst for industrial biocatalysis. However, the engineering of R. ruber has been hampered by the lack of efficient genome editing tools. This study aims at developing efficient genome editing methods for engineering R. ruber into superior strains for acrylamide production and into universal host strains for heterologous enzyme expression. CRISPR/Cas9-based counterselection was developed in R. ruber by introducing cas9 gene and sgRNA cassette, but the genome editing was limited by the low transformation and recombination efficiencies of R. ruber. Through the bypass of restriction-modification systems of R. ruber, the transformation efficiency was enhanced by 89-fold. By introducing bacteriophage recombinases Che9c60 and Che9c61, the homologous recombination efficiency of R. ruber was improved significantly. Afterward, a CRISPR/Cas9-based ssDNA and dsDNA recombineering system was developed for R. ruber, enabling successful gene deletion, insertion and mutation with an editing efficiency up to 75%. By optimizing the expression level of Cas9 protein and screening a novel recombinase RrRecT, the CRISPR/Cas9-based ssDNA recombineering system was also successfully applied to another species, R. opacus. Homologous double-crossover based on sacB-counterselection is a simple and fast technique for genome editing. However, the originial method failed to work for R. ruber because of the low integration efficiency of suicide plasmids, high false positive rate and ineffectiveness of sacB. The antibiotic resistance marker of the suicide plasmids was revealed to a critical factor and optimized to reduce the false positive rate. The expression of sacB was enhanced to effectively confer sucrose sensitivity in R. ruber. The restriction-modification systems of R. ruber were analyzed and bypassed to improve the integration efficiency of the suicide plasmids. Based on the above modifications, an optimized sacB-based double-crossover method was developed, which enabled efficient genome editing of R. ruber, including the deletion of large DNA fragments up to 46.5 kb. Compared with the CRISPR/Cas9 system, this method shortens the period of singe gene deletion from 21 days to 7 days.R. ruber was then engineered by genome editing to construct superior strains for acrylamide production. The formation of byproduct acrylic acid was totally blocked by analyzing and deleting the critical amidase genes of R. ruber. The catalytic stability of Rhodococcus cells was significantly enhanced by in-situ mutating the nitrile hydratase gene into a stable mutant. The catalytic efficiency was improved by more than 40% through the deletion of regulator genes. Four engineered R. ruber strains with superior performance were constructed and successfully used for industrial-scale production of acrylamide, which significantly reduced energy and material consumption.Furthermore, R. ruber was engineered into an efficient host strain for the heterologous expression of different enzymes including epoxide hydrolase, nitrilase and transaminase. The expression efficiency and catalytic stability of epoxide hydrolase in recombinant Escherichia coli and R. ruber was assessed and compared. The epoxide hydrolase in R. ruber accumulated up to 30% of the total soluable intracellular proteins, and showed better thermal stability, pH stability, and substrate and product tolerance than the enzyme in recombinant E. coli. The cloning and expression of nitrilase and transaminase further confirmed the universality of R. ruber as efficient and stable whole-cell biocatalysts. Strategies for engineering R. ruber into more efficient hosts were also proposed and examined.