Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Abstract

SummaryReporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green¯uorescent protein (GFP) de novo, optimizing its codon usage to re¯ect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5¢-and 3¢-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a nonoptimized GFP cassette (GFPncb), also under the control of the rbcL 5¢-and 3¢-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate »80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5¢-and 3¢-UTRs, is suf®ciently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Northern blots indicated that the increase of codon-optimized CNTB-FVIII HC and CNTB-VP1 accumulation is at the translational level rather than RNA transcript accumulation. Several previous studies on the expression of foreign genes have shown a lack of variation or modest increases in transcript abundance but significant variation in translation efficiency (Franklin et al, 2002;Gisby et al, 2011;Nakamura et al, 2016). Franklin et al (2002) reported a lack of variation in transcript abundance for GFP expression in Chlamydomonas reinhardtii chloroplasts despite an 80-fold increase in GFP protein accumulation of the codonoptimized sequence.…”

“…Several previous studies on the expression of foreign genes have shown a lack of variation or modest increases in transcript abundance but significant variation in translation efficiency (Franklin et al, 2002;Gisby et al, 2011;Nakamura et al, 2016). Franklin et al (2002) reported a lack of variation in transcript abundance for GFP expression in Chlamydomonas reinhardtii chloroplasts despite an 80-fold increase in GFP protein accumulation of the codonoptimized sequence. Even though there was a 3-fold increase in mRNA levels of codon-optimized TGF-b3 when compared with the native sequence (Gisby et al, 2011), the greater part of the 75-fold increase in synthetic TGF-b sequence was attributed to enhanced translation.…”

“…However, human TGF-b3 (13 kD, 56% GC) was expressed in up to 12% of leaf protein only after codon optimization (Gisby et al, 2011). Also, the expression of GFP (approximately 26 kD) increased approximately 80-fold after codon optimization (Franklin et al, 2002). Therefore, proteins with shorter coding sequences are not ideal to evaluate codon optimization concepts and other limitations in translation.…”

“…In vitro assays of inserted genes with several synonymous codons show that translation efficiency does not always correlate with codon usage in plastid mRNAs (Nakamura and Sugiura, 2007), but they have been used in several codon optimization studies (Lutz et al, 2001;Ye et al, 2001;Franklin et al, 2002;Lenzi et al, 2008;Jabeen at al., 2010;Madesis et al, 2010;Gisby et al, 2011;Wang et al, 2015b;Boehm et al, 2016;Nakamura et al, 2016). While some studies achieved significant increases in expression (75-to 80-fold) after codon optimization (Franklin et al, 2002;Gisby et al, 2011), other studies observed negligible enhancement (Ye et al, 2001;Lenzi et al, 2008;Daniell et al, 2009;Wang et al, 2015b;Nakamura et al, 2016). However, translation initiation and the elongation efficiency of codon-optimized sequences were enhanced when chloroplast gene N-terminal sequences were inserted downstream of 5ʹ UTRs (Ye et al, 2001;Lenzi et al, 2008).…”

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (HGAD65)

“…The native hGAD65 DNA contains relatively low AT content (52%) and its expression may not be favored by an AT-rich C. reinhardtii chloroplast genome in which the overall AT content is 65.5% [43]. Franklin et al [44] showed an 80-fold increase in expression levels of GFP (green fluorescent protein) in C. reinhardtii chloroplasts when a synthetic GFP gene with increased AT content (66% AT content in synthetic GFP in comparison to native GFP with 60% AT content) was used. Furthermore, as the 5'-UTR region of chloroplast mRNAs has a profound effect on the translational efficiency of C. reinhardtii chloroplast genes [45,46], the use of different 5'-UTRs of chloroplast genes such as the 5'-UTR of the plastid psbA gene (D1 protein of photosystem II), combined with a strong promoter, could serve as another approach to enhance hGAD65 expression in C. reinhardtii cells.…”