Biosystem design of Corynebacterium glutamicum for bioproduction.

Corynebacterium glutamicum, a natural glutamate-producing bacterium adopted for industrial production of amino acids, has been extensively explored recently for high-level biosynthesis of amino acid derivatives, bulk chemicals such as organic acids and short-chain alcohols, aromatics, and natural products, including polyphenols and terpenoids. Here, we review the recent advances with a focus on biosystem design principles, metabolic characterization and modeling, omics analysis, utilization of nonmodel feedstock, emerging CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) tools for Corynebacterium strain engineering, biosensors, and novel strains of C. glutamicum. Future research directions for developing C. glutamicum cell factories are also discussed.

Production and excretion of astaxanthin by engineered Yarrowia lipolytica using plant oil as both the carbon source and the biocompatible extractant.

This study aimed to develop a bioprocess using plant oil as the carbon source for lipid-assimilating yeast to produce high-value astaxanthin. Using high-oleic safflower oil as a model, efficient cell growth and astaxanthin production by the engineered Yarrowia lipolytica strain ST7403 was demonstrated, and a considerable portion of astaxanthin was found excreted into the spent oil. Astaxanthin was the predominant carotenoid in the extracellular oil phase that allowed facile in situ recovery of astaxanthin without cell lysis. Autoclaving the safflower oil medium elevated the peroxide level but it declined quickly during fermentation (reduced by 84% by day 3) and did not inhibit cell growth or astaxanthin production. In a 1.5-L fed-batch bioreactor culture with a YnB-based medium containing 20% safflower oil, and with the feeding of casamino acids, astaxanthin production reached 54 mg/L (53% excreted) in 28 days. Further improvement in astaxanthin titer and productivity was achieved by restoring leucine biosynthesis in the host, and running fed-batch fermentation using a high carbon-to-nitrogen ratio yeast extract/peptone medium containing 70% safflower oil, with feeding of additional yeast extract/peptone, to attain 167 mg/L astaxanthin (48% excreted) in 9.5 days of culture. These findings facilitate industrial microbial biorefinery development that utilizes renewable lipids as feedstocks to not only produce high-value products but also effectively extract and recover the products, including non-native ones.Key Points• Yarrowia lipolytica can use plant oil as a C-source for astaxanthin production.• Astaxanthin is excreted and accumulated in the extracellular oil phase.• Astaxanthin is the predominant carotenoid in the extracellular oil phase.• Plant oil serves as a biocompatible solvent for in situ astaxanthin extraction. Graphical abstract.

Biosynthesis of terpene compounds using the non-model yeast Yarrowia lipolytica: grand challenges and a few perspectives.

Yarrowia lipolytica has emerged as an important non-model host for terpene production. However, three main challenges remain in industrial production using this yeast. First, considerable knowledge gaps exist in metabolic flux across multiple compartments, cofactor generation, and catabolism of non-sugar carbon sources. Second, many enzymatic steps in the complex-terpene synthesis pathway can pose rate-limitations, causing accumulation of toxic intermediates and increased metabolic burdens. Third, metabolic shifts, morphological changes, and genetic mutations are poorly characterized under industrial fermentation conditions. To overcome these challenges, systems metabolic analysis, protein engineering, novel pathway engineering, model-guided strain design, and fermentation optimization have been attempted with some successes. Further developments that address these challenges are needed to advance the Yarrowia lipolytica platform for industrial-scale production of high-value terpenes, including those with highly complex structures such as anticancer molecules withanolides and insecticidal limonoids.

An ancient Chinese wisdom for metabolic engineering: Yin-Yang.

In ancient Chinese philosophy, Yin-Yang describes two contrary forces that are interconnected and interdependent. This concept also holds true in microbial cell factories, where Yin represents energy metabolism in the form of ATP, and Yang represents carbon metabolism. Current biotechnology can effectively edit the microbial genome or introduce novel enzymes to redirect carbon fluxes. On the other hand, microbial metabolism loses significant free energy as heat when converting sugar into ATP; while maintenance energy expenditures further aggravate ATP shortage. The limitation of cell "powerhouse" prevents hosts from achieving high carbon yields and rates. Via an Escherichia coli flux balance analysis model, we further demonstrate the penalty of ATP cost on biofuel synthesis. To ensure cell powerhouse being sufficient in microbial cell factories, we propose five principles: 1. Take advantage of native pathways for product synthesis. 2. Pursue biosynthesis relying only on pathways or genetic parts without significant ATP burden. 3. Combine microbial production with chemical conversions (semi-biosynthesis) to reduce biosynthesis steps. 4. Create "minimal cells" or use non-model microbial hosts with higher energy fitness. 5. Develop a photosynthesis chassis that can utilize light energy and cheap carbon feedstocks. Meanwhile, metabolic flux analysis can be used to quantify both carbon and energy metabolisms. The fluxomics results are essential to evaluate the industrial potential of laboratory strains, avoiding false starts and dead ends during metabolic engineering.

Photoheterotrophic fluxome in Synechocystis sp. strain PCC 6803 and its implications for cyanobacterial bioenergetics.

This study investigated metabolic responses in Synechocystis sp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O2 evolution and ATP/NADPH generation by linear electron flow. Based on (13)C-metabolic flux analysis ((13)C-MFA) and RNA sequencing, we have found that Synechocystis sp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO2 and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.

Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis.

This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields - the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route - in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories.

Central metabolic responses to the overproduction of fatty acids in Escherichia coli based on 13C-metabolic flux analysis.

We engineered a fatty acid overproducing Escherichia coli strain through overexpressing tesA ("pull") and fadR ("push") and knocking out fadE ("block"). This "pull-push-block" strategy yielded 0.17 g of fatty acids (C12­C18) per gram of glucose (equivalent to 48% of the maximum theoretical yield) in batch cultures during the exponential growth phase under aerobic conditions. Metabolic fluxes were determined for the engineered E. coli and its control strain using tracer ([1,2-13C]glucose) experiments and 13C-metabolic flux analysis. Cofactor (NADPH) and energy (ATP) balances were also investigated for both strains based on estimated fluxes. Compared to the control strain, fatty acid overproduction led to significant metabolic responses in the central metabolism: (1) Acetic acid secretion flux decreased 10-fold; (2) Pentose phosphate pathway and Entner­Doudoroff pathway fluxes increased 1.5- and 2.0-fold, respectively; (3) Biomass synthesis flux was reduced 1.9-fold; (4) Anaplerotic phosphoenolpyruvate carboxylation flux decreased 1.7-fold; (5) Transhydrogenation flux converting NADH to NADPH increased by 1.7-fold. Real-time quantitative RT-PCR analysis revealed the engineered strain increased the transcription levels of pntA (encoding the membrane-bound transhydrogenase) by 2.1-fold and udhA (encoding the soluble transhydrogenase) by 1.4-fold, which is in agreement with the increased transhydrogenation flux. Cofactor and energy balances analyses showed that the fatty acid overproducing E. coli consumed significantly higher cellular maintenance energy than the control strain. We discussed the strategies to future strain development and process improvements for fatty acid production in E. coli.

Optimize flue gas settings to promote microalgae growth in photobioreactors via computer simulations.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions(1). Microalgae not only capture solar energy more efficiently than plants(3), but also synthesize advanced biofuels(2-4). Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth(5). On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient "flue gas to algae" system. Researchers have proposed different photobioreactor configurations(4,6) and cultivation strategies(7,8) with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Role of dopant concentration, crystal phase and particle size on microbial inactivation of Cu-doped TiO2 nanoparticles.

The properties of Cu-doped TiO(2) nanoparticles (NPs) were independently controlled in a flame aerosol reactor by varying the molar feed ratios of the precursors, and by optimizing temperature and time history in the flame. The effect of the physico-chemical properties (dopant concentration, crystal phase and particle size) of Cu-doped TiO(2) nanoparticles on inactivation of Mycobacterium smegmatis (a model pathogenic bacterium) was investigated under three light conditions (complete dark, fluorescent light and UV light). The survival rate of M. smegmatis (in a minimal salt medium for 2 h) exposed to the NPs varied depending on the light irradiation conditions as well as the dopant concentrations. In dark conditions, pristine TiO(2) showed insignificant microbial inactivation, but inactivation increased with increasing dopant concentration. Under fluorescent light illumination, no significant effect was observed for TiO(2). However, when TiO(2) was doped with copper, inactivation increased with dopant concentration, reaching more than 90% (>3 wt% dopant). Enhanced microbial inactivation by TiO(2) NPs was observed only under UV light. When TiO(2) NPs were doped with copper, their inactivation potential was promoted and the UV-resistant cells were reduced by over 99%. In addition, the microbial inactivation potential of NPs was also crystal-phase-and size-dependent under all three light conditions. A lower ratio of anatase phase and smaller sizes of Cu-doped TiO(2) NPs resulted in decreased bacterial survival. The increased inactivation potential of doped TiO(2) NPs is possibly due to both enhanced photocatalytic reactions and leached copper ions.

Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris hildenborough using gas chromatography-mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry.

Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.