Mamta Gupta , Matthew Wong , Kamran Jawed , Kamil Gedeon , Hannah Barrett , Marcelo Bassalo , Clifford Morrison , Danish Eqbal , Syed Shams Yazdani , Ryan T. Gill , Jiaqi Huang , Marc Douaisi , Jonathan Dordick , Georges Belfort , Mattheos A.G. Koffas
{"title":"Isobutanol production by combined in vivo and in vitro metabolic engineering","authors":"Mamta Gupta , Matthew Wong , Kamran Jawed , Kamil Gedeon , Hannah Barrett , Marcelo Bassalo , Clifford Morrison , Danish Eqbal , Syed Shams Yazdani , Ryan T. Gill , Jiaqi Huang , Marc Douaisi , Jonathan Dordick , Georges Belfort , Mattheos A.G. Koffas","doi":"10.1016/j.mec.2022.e00210","DOIUrl":null,"url":null,"abstract":"<div><p>The production of the biofuel, isobutanol, in <em>E. coli</em> faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both <em>in vivo</em> with <em>in vitro</em> metabolic engineering to produce isobutanol. The <em>in vivo</em> production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in <em>E. coli</em> and inhibition of competitive valine pathway using CRISPRi technology. The subsequent <em>in vitro</em> conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the <em>in vivo</em> production of KIV and subsequent <em>in vitro</em> production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L<sup>−1</sup> h<sup>−1</sup>, and titers of 5.6 and 1.78 g L<sup>−1</sup>, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with <em>in vivo</em> production using a consolidated bioprocess.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030122000190/pdfft?md5=b291a3d0e60096f33c80f32a13d96aa2&pid=1-s2.0-S2214030122000190-main.pdf","citationCount":"2","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2214030122000190","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 2
Abstract
The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L−1 h−1, and titers of 5.6 and 1.78 g L−1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.
在大肠杆菌中生产生物燃料异丁醇面临着酒精毒性、产品抑制、产品回收和长期工业可行性的限制。在这里,我们展示了一种结合体内和体外代谢工程来生产异丁醇的方法。α-酮异戊酸(KIV)的体内生成是通过CRISPR介导的大肠杆菌双胞设计(BCD)中KIV通路的整合和CRISPRi技术对竞争缬氨酸通路的抑制来实现的。随后用2-酮酸脱羧酶(KIVD)和醇脱氢酶(ADH)工程酶进行体外异丁醇转化。对于KIV的体内生产和随后的体外异丁醇生产,这种两步连续方法的产率分别为56%和93%,产率分别为0.62和0.074 g L−1 h−1,滴度分别为5.6和1.78 g L−1。因此,这种组合的生物合成系统可以作为一种模块化的方法来生产重要的代谢物,如异丁醇,而不受体内使用统一生物过程生产的限制。
期刊介绍:
Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.