Microbes have developed a multitude of possibilities to utilize CO2 as carbon source. While the CO2 fixation pathways are optimized through evolution, the production of valuable products thereof is rarely a natural trait. To efficiently use autotrophic microbes in industrial biotechnology, metabolic engineering can contribute to further yield and to rate improvement of CO2 fixation as well as to the exploitation of microbial synthesis capacities. The optimized metabolic engineering workflow includes consecutive design-built-test cycles. The computational design of metabolic networks built on genome sequencing data can direct strain engineering and thereby reduce experimental work. With growing numbers of available gene editing methods and expanding molecular toolkits, the construction of genetically engineered organisms greatly accelerated. The development of high-throughput screening systems for clone identification and phenotyping is a prerequisite to keep pace with the progress in cell factory design. The herein described metabolic engineering techniques will greatly expand the potential of autotrophic microbes in industrial biotechnology processes.