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Engineering a cyanobacterial cell factory for production of lactic acid.

ABSTRACT: Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO(2) has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an l-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to l-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

SUBMITTER: Angermayr SA

PROVIDER: S-EPMC3457509 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Engineering a cyanobacterial cell factory for production of lactic acid.

Angermayr S Andreas SA Paszota Michal M Hellingwerf Klaas J KJ

Applied and environmental microbiology 20120803 19

Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO(2) has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an l-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to l-lactic acid production, a phenotype ...[more]

PMID: 22865063

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Project description:BackgroundBifidobacteria are gram-positive, probiotic, and generally regarded as safe bacteria. Techniques such as transformation, gene knockout, and heterologous gene expression have been established for Bifidobacterium, indicating that this bacterium can be used as a cell factory platform. However, there are limited previous reports in this field, likely because of factors such as the highly anaerobic nature of this bacterium. Bifidobacterium adolescentis is among the most oxygen-sensitive Bifidobacterium species. It shows strain-specific gamma-aminobutyric acid (GABA) production. GABA is a potent bioactive compound with numerous physiological and psychological functions. In this study, we investigated whether B. adolesentis could be used for mass production of GABA.ResultsThe B. adolescentis 4-2 strain isolated from a healthy adult human produced approximately 14 mM GABA. It carried gadB and gadC, which encode glutamate decarboxylase and glutamate GABA antiporter, respectively. We constructed pKKT427::Pori-gadBC and pKKT427::Pgap-gadBC plasmids carrying gadBC driven by the original gadB (ori) and gap promoters, respectively. Recombinants of Bifidobacterium were then constructed. Two recombinants with high production abilities, monitored by two different promoters, were investigated. GABA production was improved by adjusting the fermentation parameters, including the substrate concentration, initial culture pH, and co-factor supplementation, using response surface methodology. The optimum initial cultivation pH varied when the promoter region was changed. The ori promoter was induced under acidic conditions (pH 5.2:4.4), whereas the constitutive gap promoter showed enhanced GABA production at pH 6.0. Fed-batch fermentation was used to validate the optimum fermentation parameters, in which approximately 415 mM GABA was produced. The conversion ratio of glutamate to GABA was 92-100%.ConclusionWe report high GABA production in recombinant B. adolescentis. This study provides a foundation for using Bifidobacterium as a cell factory platform for industrial production of GABA.

Project description:l-Lactic acid (LA) is a three-carbon hydroxycarboxylic acid with extensive applications in food, cosmetic, agricultural, pharmaceutical, and bioplastic industries. However, microbial LA production is limited by its intrinsic inefficiency of cellular metabolism. Here, pathway engineering was used to rewire the biosynthetic pathway for LA production in Saccharomyces cerevisiae by screening heterologous l-lactate dehydrogenase, reducing ethanol accumulation, and introducing a bacterial acetyl coenzyme A (acetyl-CoA) synthesis pathway. To improve its intrinsic efficiency of LA export, transporter engineering was conducted by screening the monocarboxylate transporters and then strengthening the capacity of LA export, leading to LA production up to 51.4 g/L. To further enhance its intrinsic efficiency of acid tolerance, adaptive evolution was adopted by cultivating yeast cells with a gradual increase in LA levels during 12 serial subcultures, resulting in a 17.5% increase in LA production to 60.4 g/L. Finally, the engineered strain S.c-NO.2-100 was able to produce 121.5 g/L LA, with a yield of up to 0.81 g/g in a 5-L batch bioreactor. The strategy described here provides a guide for developing efficient cell factories for the production of the other industrially useful organic acids. IMPORTANCE Saccharomyces cerevisiae is one of the most widely engineered cell factories for the production of organic acids. However, microbial production of l-lactic acid is limited by its intrinsic inefficiency of cellular metabolism in S. cerevisiae. Here, the transmission efficiency of the biosynthetic pathway was improved by pathway optimization to increase l-lactic acid production. Then, the synthetic ability for l-lactic acid was further enhanced by adaptive evolution to improve acid tolerance of S. cerevisiae. Based on these strategies, the final engineered S. cerevisiae strain achieved high efficiency of l-lactic acid production. These findings provide new insight into improving the intrinsic efficiency of cellular metabolism and will help to construct superior industrial yeast strains for high-level production of other organic acids.

Project description: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.

Project description:BackgroundAcyl-(acyl carrier protein (ACP)) reductase (AAR) is a key enzyme for hydrocarbon biosynthesis in cyanobacteria, reducing fatty acyl-ACPs to aldehydes, which are then converted into hydrocarbons by aldehyde-deformylating oxygenase (ADO). Previously, we compared AARs from various cyanobacteria and found that hydrocarbon yield in Escherichia coli coexpressing AAR and ADO was highest for AAR from Synechococcus elongatus PCC 7942 (7942AAR), which has high substrate affinity for 18-carbon fatty acyl-ACP, resulting in production of mainly heptadecene. In contrast, the hydrocarbon yield was lowest for AAR from Synechococcus sp. PCC 7336 (7336AAR), which has a high specificity for 16-carbon substrates, leading to production of mainly pentadecane. However, even the most productive AAR (7942AAR) still showed low activity; thus, residues within AAR that are nonconserved, but may still be important in hydrocarbon production need to be identified to engineer enzymes with improved hydrocarbon yields. Moreover, AAR mutants that favor shorter alkane production will be useful for producing diesel fuels with decreased freezing temperatures. Here, we aimed to identify such residues and design a highly productive and specific enzyme for hydrocarbon biosynthesis in E. coli.ResultsWe introduced single amino acid substitutions into the least productive AAR (7336AAR) to make its amino acid sequence similar to that of the most productive enzyme (7942AAR). From the analysis of 41 mutants, we identified 6 mutations that increased either the activity or amount of soluble AAR, leading to a hydrocarbon yield improvement in E. coli coexpressing ADO. Moreover, by combining these mutations, we successfully created 7336AAR mutants with ~ 70-fold increased hydrocarbon production, especially for pentadecane, when compared with that of wild-type 7336AAR. Strikingly, the hydrocarbon yield was higher in the multiple mutants of 7336AAR than in 7942AAR.ConclusionsWe successfully designed AAR mutants that, when coexpressed with ADO in E. coli, are more highly effective in hydrocarbon production, especially for pentadecane, than wild-type AARs. Our results provide a series of highly productive AARs with different substrate specificities, enabling the production of a variety of hydrocarbons in E. coli that may be used as biofuels.

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Engineering a cyanobacterial cell factory for production of lactic acid.

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Engineering a cyanobacterial cell factory for production of lactic acid.

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