Metabolic engineering for the production of biofuel by Rhodococcus opacus PD630

Abstract

Microbial production of fatty acids and biodiesel from non-food renewable biomass has been attracting much attention. There have been several reports on microbial production of fatty acid ethyl ester (FAEE) biodiesel from glucose, but so far the titers have been rather low (<1.5 g/liter). This is mainly due to the tightly regulated fatty acid metabolism. On the other hand, the oleaginous bacterium Rhodococcus opacus PD630 is capable of accumulating triacylglycerols (TAGs) intracellularly up to 88 % of its dry cell weight. Here we report the high-level production of free fatty acids and biodiesel by metabolic engineering of R. opacus PD630. First, culture conditions, including pH, were optimized to produce $TAG^m$ to the highest concentration (119.2 g/liter) to date from glucose. For the production of free fatty acids, R. opacus ROP1 was developed by deleting six major fadD genes (encoding fatty acyl-CoA synthetase) and amplifying two native monoacylglycerol lipases (LPD01036 and LPD02672) under the acetamide-inducible promoter. Then, plasmid pROP1_34 harboring the native TAG lipase gene (LPD05381) without its signal sequence and Burkholderia cepacia lipase specific-foldase was introduced into the ROP1 strain. Fed-batch culture of the resulting strain produced 50.2 g/liter of free fatty acids from glucose, the highest level reported to date. For the production of FAEE, R. opacus ROP3_03 was developed by deleting seven major fadE genes (encoding acyl-CoA dehydrogenase), together with replacing the promoters of the most expressed fadD gene (LPD05217) and two native monogacylglycerol lipase genes with an acetamide-inducible promoter. Next, plasmid pROP3_03, which is pROP1_34 additionally harboring an aerobically active mutant aldehyde/alcohol dehydrogenase gene from Escherichia coli and a wax ester synthase gene from Marinobacterium hydrocarbonoclasticus with optimized RBS sequences, was introduced into the ROP3 strain. Fed-batch culture of the resulting ROP3_03 strain on glucose produced 21.3 g/liter of FAEEs. For the production of long-chain hydrocarbons (LCHC), pROPA1 harboring the genes encoding Clostridium kluyveri acyl-CoA reductase, codon-optimized Synechococcus elongatus acyl-ACP reductase and decarbonylase based on pROP1_34, was constructed. Fed-batch culture of ROPA2 strain (monooxygenase-deleted ROP3 strain) harboring pROPA1 on glucose produced 5.2 g/liter of LCHC. The metabolically engineered R. opacus strains developed here will be useful as platform strains for efficient production of fatty acids and their derivative chemicals and biofuels. In addition, for the extension of metabolic engineering tool in Rhodococcus species, we constructed modified sRNAs system in R. opacus PD630. Even though a basic component was similar to E. coli system, we tested several Hfq proteins to test binding scaffold and downregulation of proteins. By applying this system, we demonstrate enhanced chemical production in R. opacus PD630, showing its suitability for the development of biorefinery.