Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth

Abstract

Chloroplasts offer high-level transgene expression and transgene containment due to maternal inheritance, and are ideal hosts for biopharmaceutical biosynthesis via multigene engineering. To exploit these advantages, we have expressed 12 enzymes in chloroplasts for the biosynthesis of artemisinic acid (precursor of artemisinin, antimalarial drug) in an alternative plant system. Integration of transgenes into the tobacco chloroplast genome via homologous recombination was confirmed by molecular analysis, and biosynthesis of artemisinic acid in plant leaf tissues was detected with the help of 13C NMR and ESI-mass spectrometry. The excess metabolic flux of isopentenyl pyrophosphate generated by an engineered mevalonate pathway was diverted for the biosynthesis of artemisinic acid. However, expression of megatransgenes impacted the growth of the transplastomic plantlets. By combining two exogenous pathways, artemisinic acid was produced in transplastomic plants, which can be improved further using better metabolic engineering strategies for commercially viable yield of desirable isoprenoid products.

Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest

“…The production was greater when the two enzymes for squalene production, squalene synthase and farnesyl diphosphate synthase, were targeted to the chloroplast. Even though the expression of the transgenes was expected to be restricted to trichomes, those transgenic plants that expressed squalene at the highest level displayed strong phenotypes such as dwarfism and chlorosis, which are similar to the phenotypes observed upon ubiquist expression of transgenes impacting the pool of IPP precursors (Wu et al, 2006;Saxena et al, 2014;Gwak et al, 2017).…”

“…In this case, production of the saponin led to a severe phenotype, including dwarfism, change in flower and pollen morphology, and impaired seed production (Gwak et al, 2017). Similarly, dwarfism was observed upon metabolic engineering of tobacco chloroplast to produce artemisinic acid (Saxena et al, 2014). All these approaches used ubiquist promoters to drive expression of the transgenes.…”

The Engineered Chloroplast Genome Just Got Smarter

“…To date, one of the most commonly used site of transgene integration is the transcriptionally active intergenic region between the trn I- trn A genes (in the rrn operon), located within the IR regions of the chloroplast genome [12, 13, 15–17], although several other sites have been explored (Figure 1B). With insertion of seven transgenes at this site, up to thirteen genes could be driven by two endogenous 16S rrn and psbA promoters [18, 19]. This flanking sequence includes the chloroplast origin of replication (that provides more copies of templates for integration) and copy correction mechanism existing within the inverted repeat regions enhance homoplasmy (see Glossary) [20]; in addition, introns present within these genes facilitate efficient processing of transgene transcripts.…”

“…In addition, an intercistronic expression element (IEE) was introduced into the spacer region between cistrons to enhance processing of polycistrons into monocistrons to enhance translation [7]. However, this concept contradicts recent in depth ribosome profiling studies [30] that show similar translation efficiency in both spliced and unspliced native chloroplast polycistrons, as observed previously by high level expression of several heterologous polycistrons via the chloroplast genome without IEE [21, 31] or multigenes engineered recently via the chloroplast genome [18, 19]. New PCR methods using overlapping primers has been used to eliminate introns and splice exons facilitate expression of eukaryotic genes without the need for cDNA libraries; this concept was successfully employed to transform the chloroplast genome with fungal genes containing >10 introns [32].…”

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

“…As results, three kinds of artemisinin precursors including amorpha-1,4-diene, artemisinic alcohol, and dihydroartemisinic alcohol were produced, but no artemisinin was detectable . Recently, as many as 12 kinds of artemisinin biosynthesis genes were introduced into the plastid genome of N. tabacum, resulting in the accumulation of artemisinic acid, but still no artemisinin was found (Saxena et al 2014).…”