Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803

Abstract

BackgroundMolecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by “designer microbes.” The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make “designer microbes” includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made.ResultsTo improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme’s affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization.ConclusionAn efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.

Advances in proteomics for production strain analysis

“…Synechocystis is a fully-sequenced, naturally transformable strain of cyanobacterium that is gaining popularity as a model production chassis [23]. It is currently being investigated for a variety of different products including precursors such as isoprenoids and lactic acid from CO 2 [24,25], as well as biofuels like ethanol, butanol and hydrogen .…”

Avaliação Dos Metabólitos Produzidos Por Fotofermentação Por Cianobactérias

“…Entretanto, os ácidos orgânicos não são sintetizados em grande quantidade pelas cianobactérias, devido à utilização do carbono fixado para a manutenção celular (ANGERMAYR et al, 2014). Com relação aos biocombustíveis, que são uma das alternativas para suprir a escassez futura de recursos fósseis, pois são advindos de fontes renováveis e apresentam baixos custos de produção, o etanol pode ser sintetizado pelas cianobactérias, o qual é um subproduto do ciclo do Calvin (HALLENBECK, 2012).…”

An overview on biofuel and biochemical production by photosynthetic microorganisms with understanding of the metabolism and by metabolic engineering together with efficient cultivation and downstream processing

“…Since Pyk is inhibited under light condition in Synechosystis, heterologous expression of Pyk can enhance the pyruvate production and in turn enhance the lactate production, where Pyk is allosterically activated by fructose 1,6-bisphosphate (FBP) in the case of Pyk-F originated form E. coli, while the original Pyk does not show such characteristics [136]. Moreover, Ppc may be knocked down to direct the carbon flow from PEP towards lactate production via PYR, but the cell growth is depressed, since Ppc is also an important pathway for CO 2 fixation [136].…”

“…Since Pyk is inhibited under light condition in Synechosystis, heterologous expression of Pyk can enhance the pyruvate production and in turn enhance the lactate production, where Pyk is allosterically activated by fructose 1,6-bisphosphate (FBP) in the case of Pyk-F originated form E. coli, while the original Pyk does not show such characteristics [136]. Moreover, Ppc may be knocked down to direct the carbon flow from PEP towards lactate production via PYR, but the cell growth is depressed, since Ppc is also an important pathway for CO 2 fixation [136]. In most bacteria, LDH requires cofactor NADH, whereas NADPH is abundant in Synechosystis as mentioned before, and thus NADPH-dependent LDH may increase the lactate production, where this may be partly attained by introducing the LDH from B. subtilis, of which LDH co-utilizes NADH and NADPH [136,137] (Table 1).…”

“…Lactic acid has been used in the food and pharmaceutical industries and for biodegradable polymers [134], and this can be produced in Synechosystis after heterologous expression of LDH [135,136]. Since Pyk is inhibited under light condition in Synechosystis, heterologous expression of Pyk can enhance the pyruvate production and in turn enhance the lactate production, where Pyk is allosterically activated by fructose 1,6-bisphosphate (FBP) in the case of Pyk-F originated form E. coli, while the original Pyk does not show such characteristics [136].…”