Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound

Abstract

Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.

Cyanobacterial Production of Biopharmaceutical and Biotherapeutic Proteins

“…However, recent experience has also shown that heterologous expression of eukaryotic plant and yeast genes occurs at low protein levels, regardless of the promoter used and mRNA levels achieved in the cyanobacterial cytosol (Formighieri and Melis, 2016). For example, plant terpene synthases could not be expressed well in cyanobacteria under the control of different strong endogenous and heterologous promoters (Formighieri and Melis, 2014;Englund et al, 2018). Heterologous expression in cyanobacteria of the isoprene synthase (Lindberg et al, 2010;Bentley and Melis, 2012), β-phellandrene synthase (Bentley et al, 2013), geranyl diphosphate (GPP) synthase from a higher plant origin (Bentley et al, 2014;Formighieri and Melis, 2017;Betterle and Melis, 2018), and the alcohol dehydrogenase (ADH1) gene from yeast (Chen and Melis, 2013), all showed low levels of recombinant protein expression, even under the control of strong endogenous (e.g., psbA2, rbcL, cpc) or strong heterologous promoters (e.g., Ptrc), and even after following a careful codonuse optimization of the target transgene (Lindberg et al, 2010;Bentley and Melis, 2012;Ungerer et al, 2012;Bentley et al, 2013;Chen and Melis, 2013;Formighieri and Melis, 2014;Englund et al, 2018).…”

“…For example, plant terpene synthases could not be expressed well in cyanobacteria under the control of different strong endogenous and heterologous promoters (Formighieri and Melis, 2014;Englund et al, 2018). Heterologous expression in cyanobacteria of the isoprene synthase (Lindberg et al, 2010;Bentley and Melis, 2012), β-phellandrene synthase (Bentley et al, 2013), geranyl diphosphate (GPP) synthase from a higher plant origin (Bentley et al, 2014;Formighieri and Melis, 2017;Betterle and Melis, 2018), and the alcohol dehydrogenase (ADH1) gene from yeast (Chen and Melis, 2013), all showed low levels of recombinant protein expression, even under the control of strong endogenous (e.g., psbA2, rbcL, cpc) or strong heterologous promoters (e.g., Ptrc), and even after following a careful codonuse optimization of the target transgene (Lindberg et al, 2010;Bentley and Melis, 2012;Ungerer et al, 2012;Bentley et al, 2013;Chen and Melis, 2013;Formighieri and Melis, 2014;Englund et al, 2018). Similarly, only low levels of expression were reported for a chimeric complex of plant enzymes, including the ethylene synthase efe gene from Solanum lycopersicum (tomato) (Jindou et al, 2014;Xue and He, 2014), limonene synthase from Mentha spicata (spearmint) (Davies et al, 2014) and Picea sitchensis (Sitka spruce) (Halfmann et al, 2014b), the sesquiterpene farnesene and bisabolene synthases from Picea abies (Norway spruce) (Halfmann et al, 2014a) and Abies grandis (grand fir) (Davies et al, 2014).…”

Combinatorial metabolic engineering platform enabling stable overproduction of lycopene from carbon dioxide by cyanobacteria

“…Lycopene is produced endogenously by Synechocystis and other cyanobacteria as a precursor of other carotenoids used for photoprotection, but does not naturally accumulate ( Figure 2). To overproduce lycopene a pathway-encoding construct was designed using homologs of three native CDSs ( dxs , crtE , and crtB ) to boost pathway flux ( dxs is a known rate-limiting reaction in both E. coli 37,63,64 and cyanobacteria 14,15,65 ) and a heterologous CDS ( ctrI ), which provides a bacterial-type one-step desaturase to bypass the native photoautotroph-type two-step desaturase pathway 66 ( crtP and crtQ ) in the conversion of phytoene to lycopene ( Figure 2).…”

“…Cyanobacteria have been genetically modified to synthesise a wide range of non-native compounds ranging from commodity chemicals and biofuels to high-value products, including alcohols [5][6][7][8] , organic acids [8][9][10][11] , diols 12,13 , terpenoids [14][15][16][17][18][19][20] , sugars [21][22][23] and many others [24][25][26][27][28][29] . However, production using cyanobacteria is not yet commercially competitive [30][31][32] with production by petrochemical or conventional heterotrophic biotechnology approaches, both of which benefit from the current low economic costs of using fossil carbon and emitting CO 2 .…”

High density cultivation for efficient sesquiterpenoid biosynthesis inSynechocystissp. PCC 6803

“…PCC 6803 (from here: Synechocystis) resulted in 2.5-fold higher isoprene yields [12]. Likewise, numerous rational design studies targeting selected bottleneck steps within the MEP pathway as well as selected steps of upstream carbon fluxes lent support to this concept [8,9,[13][14][15][16][17][18][19]. Further strategies sought to challenge the problem of inherently weak terpene synthase activities by increasing cellular enzyme titers, concurrently pushing carbon flux towards the heterologous metabolic sink.…”