Combinatorial use of environmental stresses and genetic engineering to increase ethanol titres in cyanobacteria

Abstract

Current industrial bioethanol production by yeast through fermentation generates carbon dioxide. Carbon neutral bioethanol production by cyanobacteria uses biological fixation (photosynthesis) of carbon dioxide or other waste inorganic carbon sources, whilst being sustainable and renewable. The first ethanologenic cyanobacterial process was developed over two decades ago using Synechococcus elongatus PCC 7942, by incorporating the recombinant pdc and adh genes from Zymomonas mobilis. Further engineering has increased bioethanol titres 24-fold, yet current levels are far below what is required for industrial application. At the heart of the problem is that the rate of carbon fixation cannot be drastically accelerated and carbon partitioning towards bioethanol production impacts on cell fitness. Key progress has been achieved by increasing the precursor pyruvate levels intracellularly, upregulating synthetic genes and knocking out pathways competing for pyruvate. Studies have shown that cyanobacteria accumulate high proportions of carbon reserves that are mobilised under specific environmental stresses or through pathway engineering to increase ethanol production. When used in conjunction with specific genetic knockouts, they supply significantly more carbon for ethanol production. This review will discuss the progress in generating ethanologenic cyanobacteria through chassis engineering, and exploring the impact of environmental stresses on increasing carbon flux towards ethanol production.

Keywords: Carbon partitioning; Cyanobacteria; Environmental stress; Ethanol; Microbial pathway engineering; Synechocystis PCC 6803; Synthetic biology.

Fig. 1

Native and engineered routes from carbon dioxide to ethanol in Synechocystis. Additional pathways are shown that provide flux through pyruvate. The pathway in red is the engineered ethanologenic route from Zymomonas mobilis. Pathway intermediates: GAP, glyceraldehyde-3-phosphate; OAA, oxaloacetate; 2-OG, α-ketoglutarate; PEP, phosphoenolpyruvate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; PHB, polyhydroxybutyrate; RuBP, ribulose bisphosphate. Enzymes/genes: acc, acetyl-CoA carboxylase; acs, acetyl-CoA-synthase; ackA, acetate kinase; adh, alcohol dehydrogenase; aldDH, aldehyde dehydrogenase; eno, enolase; ldh, lactate dehydrogenase; me, malic enzyme; Pdc: pyruvate decarboxylase; pdh, pyruvate dehydrogenase complex; pepck, phosphoenolpyruvate carboxykinase; pgm, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase; phaA, acetyl-CoA acetyltransferase; phaB, acetoacetyl-CoA reductase; PhaC/E, poly(3-hydroxyalkanoate) polymerase; ppc, phosphoenolpyruvate carboxylase; pps, phosphoenolpyruvate synthase; pta, phosphate acetyltransferase; pyk, pyruvate kinase; RuBisCO, ribulose-1,5-biphosphate carboxylase/oxygenase. Lactate and ethanol are both readily secreted by Synechocystis.j

Fig. 2

CBB pathway enzymatic steps predicted to positively influence flux control [31, 70, 72, 73, 77, 78]. Pathway intermediates: DHAP, dihydroxyacetone phosphate; FBP, fructose-1,6-bisphosphate; G3P, glyceraldehyde-3-phosphate; Pi, pyrophosphate; S7P, sedoheptulose-7-phosphate; SBP, sedoheptulose-1,7-bisphosphate; Xu5P, xylulose-5-phosphate. Enzymes/genes: fba, fructose-bisphosphate aldolase; SBPase, sedoheptulose-1,7-bisphosphatase; tkl, transketolase. The remaining pathway intermediates and enzymes are defined in Fig. 1 legend. The crystal structures of RuBisCO, SBPase, tkl and fba were generated in Chimera [79] using pdb accession codes 6hbc, 3oi7, 1trk and 1rv8, respectively (https://www.rcsb.org/)

Fig. 3

Metabolic pathways from the CBB cycle towards pyruvate and glycogen production. The enzymes are shown in blue. Pathway intermediates: ADP-glc, ADP-glucose; 1,3bisPGA, 1,3-bisphosphoglycerate; aldB, α-acetolactate decarboxylase; als, acetolactate synthase; butA, butanediol dehydrogenase; F6P, fructose-6-phosphate; Gln-6P, 6-phosphogluconate; GL-6P, 6-phosphogluconolactone; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; Ru5P, ribulose-5-phosphate. Enzymes/genes: ccr, crotonyl-CoA carboxylase/reductase; glgA, glycogen synthase; glgB, glycogen branching enzyme; glgC, ADP-glucose pyrophosphorylase; PduP/MhpF, acetaldehyde dehydrogenase; phaJ/fadB, enoyl-CoA hydratase; Ter, trans-2-enoyl-CoA reductase

Fig. 4

Effect of cyanobacterial chassis engineering and environmental stress responses on bioethanol production [27, 36, 39]. The enzymes/genes are colour coded to reflect which of the three engineered chassis they belong to. Enzymes/genes: ecaA, α-type carbonic anhydrase; groESL, alcohol tolerant chaperonin; iciB, inorganic carbon transporter. The remaining intermediates and enzymes are defined in Figs. 1, 2, 3 legends