title: Tackling codon usage bias for heterologous expression in Rhodobacter sphaeroides by supplementation of rare tRNAs.
abstract: The photosynthetic Rhodobacter species are promising alternative expression hosts in bioproduction and biorefinery due to their unique metabolic capacities. With prominent inner membrane areas and efficient endogenous translocation machineries, they are especially attractive for membrane protein expression. However, codon usage bias could be a limitation in the engineering of Rhodobacter species and has seldom been investigated. In this study, we tackled the codon bias of Rhodobacter by functionally expressing 8 rare tRNAs of Rhodobacter sphaeroides with a multi-copy vector. The impact of tRNA supplementation was evaluated through monitoring expression levels of two heterologous proteins with different phylogenetic origins, a membrane subunit of the riboflavin transporter, RibU, from Lactobacillus acidophilus La-14 and a decaheme cytochrome, MtrA, from Shewanella oneidensis. Our results showed that the performances were closely related to medium composition and rare codon percentages of raw DNA sequences. Provision of rare tRNAs has increased RibU production by 7.7-folds and 2.86-fold in minimal medium and rich medium, respectively, while MtrA levels were increased by 1-fold in minimal medium. The present study confirms the presence of codon bias in R. sphaeroides and offers a facile tool for improving heterologous expression of rare-codon containing genes. We anticipate that this tRNA supplementation system can be further extended to other species of Rhodobacter, and thus will facilitate the engineering of purple bacteria for interesting applications in microbial technology.
authors.affiliation: Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors.forename: Danhui
authors.initials: D
authors.lastname: Cheng
authors.affiliation: Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors.forename: Ri
authors.initials: R
authors.lastname: Wang
authors.affiliation: Departmental of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Kristala Jones
authors.initials: KJ
authors.lastname: Prather
authors.affiliation: Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Division of Life Sciences, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
authors.forename: King Lau
authors.initials: KL
authors.lastname: Chow
authors.affiliation: Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong; Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Electronic address: kehsing@ust.hk.
authors.forename: I-Ming
authors.initials: IM
authors.lastname: Hsing
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D057005:Bioengineering; D003062:Codon / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003062:Codon; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; C100938:MtrA protein, Bacteria; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; C019079:riboflavin-binding protein; D049452:Green Fluorescent Proteins; D012343:RNA, Transfer
keywords: Bacterial expression system; Codon bias; Heterologous expression level; Rhodobacter sphaeroides; tRNA level