Modular control of multiple pathways of Corynebacterium glutamicum for 5-aminolevulinic acid production

Abstract5-aminolevulinic acid (ALA) has broad potential applications in the medical, agricultural and food industries. Several strategies have been implemented successfully to try to improve ALA synthesis. Nonetheless, the low yield has got in the way of large-scale bio-manufacture of 5-ALA. In this study, we explored strain engineering strategies for high‐level 5‐ALA production in Corynebacterium glutamicum F343 using the C4 pathway. Initially, the glutamate dehydrogenase-encoding gene gdhA was deleted to reduce glutamate yield. Then the C4 pathway was introduced in the gdhA mutant strain F2-A (∆gdhA + hemA), resulting in a 5-ALA yield of up to 3.2 g/L. Furthermore, the accumulations of downstream metabolites such as heme, porphobilinogen, and protoporphyrin IX, were decreased. After evaluating the mechanisms of this synthetic pathway by RNA-Seq, the results showed that genes involved in both the C5 pathway and heme pathways were down-regulated in strain F2-A (∆gdhA + hemA). Interestingly, upstream genes of succinyl-CoA in the tricarboxylic acid (TCA) cycle, such as icd, lpdA, were up-regulated, while its downstream genes, including sucC, sucD, sdhB, sdhA, sdhCD, were down-regulated. These changes amplify the sources of succinyl-CoA and reduce its expenditure, before pulling the carbon flux to produce 5-ALA. Furthermore, the down-regulation of most genes of the heme pathway could reduce the drainage of 5‐ALA, which further enhance its accumulation. To alleviate competition between glyoxylate and the TCA cycle, the isocitrate dehydrogenase-encoding gene aceA was also knocked out, resulting in 3.86 g/L of 5‐ALA. Finally, the fermentation conditions were optimized, resulting in a maximum 5-ALA yield of 5.6 g/L. Overall, the blocking of the glutamate synthesis pathway could be a powerful strategy to re-allocate the carbon flux to produce 5-ALA. It could also enable the efficient synthesis of other TCA derivatives in C. glutamicum.

Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli

Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to the C4 pathway. To promote PBG stability and accumulation, Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) was applied to repress hemC expression and, therefore, reduce carbon flowthrough toward porphyrin biosynthesis with minimal impact to cell physiology. To further enhance PBG biosynthesis and accumulation under the hemC-repressed genetic background, we further heterologously expressed native E. coli hemB. Using these engineered E. coli strains for bioreactor cultivation based on ~ 30 g L−1 glycerol, we achieved high PBG titers up to 209 mg L−1, representing 1.73% of the theoretical PBG yield, with improved PBG stability and accumulation. Potential biochemical, genetic, and metabolic factors limiting PBG production were systematically identified for characterization. Graphical Abstract

Impact of global warming (1.5ºC) on the productivity of selected C3 and C4 crops across Tamil Nadu

Over the last century, mean annual temperatures increased by ~1°C. UNFCCC has proposed to limit warming below 1.5°C relative to pre-industrial levels. A study was conducted on rice (C3 pathway) and maize (C4 pathway) over Tamil Nadu using DSSAT to understand the climate change impacts with projected temperature increase of 1.5°C.The future climate under RCP 4.5 and RCP 8.5 indicated 1.5°Cincrease in temperature to happen by 2053 and 2035, respectively over Tamil Nadu.Annual rainfall deviations in RCP4.5 showed drier than current condition and RCP8.5 projected wetter SWM and drier NEM (90 % of current rainfall).Impact of 1.5°C warming on crop phenology indicated 8 days reduction in duration for rice and maize. The W UE of rice would decrease by 17 per cent at current CO2 whereas, enrichment (430 ppm) would reduce by12 per cent and rice yield is reduced by 21 per cent with 360 ppm CO2 and 430 ppm reducedby 17 per cent. There is no considerable varaition (- 5 to 1 %) in maize productivity with 1.5 ºC warming. The above results indicated that 1.5 ºC warming has more negative impacts on plants with C3 compared to C4 pathway

Heterotrophic euglenid Rhabdomonas costata resembles its phototrophic relatives in many aspects of molecular and cell biology

Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.

LEAF ANATOMY CHARACTERIZATION OF FOUR Apochloa SPECIES: A C3 GENUS RELATED TO EVOLUTION OF C4 PATHWAY IN GRASSES

Leaf anatomy characteristics provide important evidences about the transition between C3 and C4 pathways. The C4 photosynthesis pathway allowed to reduce the C3 photorespiratory rate, concentrating CO2 around the Rubisco site and using structures and machinery already presented in C3 plants. In monocots, it is observed a high number of C4 lineages, most of them phylogenetically related to C3 groups. The genus Apochloa (C3), subtribe Arthropogoninae, is related to two C4 genera Coleataenia and Cyphonanthus. The aim of this study was to evaluate four Apochloa species in order to establish anatomical characteristics related to the evolution of C4 pathway in this group. By means of transverse sections fully expanded leaves of A. euprepes, A. lorea, A. molinioides, and A. poliophylla were collected and the characteristics of the mesophyll (M) and bundle sheath (BS) cells were determined. These species showed a rustic Kranz anatomy with enlarged and radial arranged BS cells, which have few organelles organized in a centrifugal position. Although the modifications of BS cells are probably related to the maintenance of plant water status, we also discuss the evolution for the establishment of C4 photosynthesis in the related C4 genera.

Expression of Photosynthesis Pathway Gene From Rice and Maize for Understanding Role in Plant Stress and Development Using Bioinformatics Approaches

Background Rice and maize go to family Poaceae contains many crops of agronomic trait and also represent two carbon metabolism systems, C3 and C4. Analysis of the maize sequence provides new insights into the employment of C3 genes to the C4 mechanism which allowed us to identify more orthologs in other crops. This investigation reports comparative account of genome wide in silico identification of C4 pathway related genes from Zea maize (Zm) and Oryza sativa (Os) from the available whole genome sequence information. The annotation of gene sequences, signature motif analysis, protein phophorylation analysis, study of upstream cis-acting elements, phylogenetic tree construction, chromosomal locations, syntenic mapping and microarray expression analysis of C4 pathway related gene family from both the genomes have been attempted. Results A total of 30 and 37 C4-pathway genes have been predicted from rice and maize genome respectively. Multiple-sequence-alignment and signature motif analysis of these proteins of rice and maize revealed high conserveness. Phophorylation analysis revealed that maize have high number than rice. The phylogenetic analysis of C4 related genes across both plant species clearly resulted in four sub-groups in both plants. In Rice, the 30 genes of C4 pathway related genes family are distributed on eleven out of twelve chromosomes, while in maize, they are randomly distributed on all the chromosomes. Most of the genes of Zm’s chromosome 1 show syntenic relationship with chromosome 1. The cis-regulatory-elements of Zm and Os genes suggested its diverse functions associated with plant growth development, stress and hormone responsiveness as well as endosperm and meristem specific gene expression. This investigation of Zm and Os can now offer new insights into the role of different C4 pathway related genes and examine the comparative syntenic mapping between two monocot models and allows for better understanding about how genes evolve within monocots. Therefore, in silico investigation of C4-photosynthetic-pathway gene family needs to be supported by wet lab experimentation of the novel genes for elucidating their function in many biological courses.Conclusion Results revealed that photosynthetic pathway related gene play a potent role in stress response and plant growth and development.

The Development of Crassulacean Acid Metabolism (CAM) Photosynthesis in Cotyledons of the C4 Species, Portulaca grandiflora (Portulacaceae)

Portulaca grandiflora simultaneously utilizes both the C4 and Crassulacean acid metabolism (CAM) photosynthetic pathways. Our goal was to determine whether CAM developed and was functional simultaneously with the C4 pathway in cotyledons of P. grandiflora. We studied during development whether CAM would be induced with water stress by monitoring the enzyme activity, leaf structure, JO2 (rate of O2 evolution calculated by fluorescence analysis), and the changes in titratable acidity of 10 and 25 days old cotyledons. In the 10 days old cotyledons, C4 and CAM anatomy were evident within the leaf tissue. The cotyledons showed high titratable acid levels but a small CAM induction. In the 25 days old cotyledons, there was a significant acid fluctuation under 7 days of water stress. The overall enzyme activity was reduced in the 10 days old plants, while in the 25 days old plants CAM activity increased under water-stressed conditions. In addition to CAM, the research showed the presence of glycine decarboxylase in the CAM tissue. Thus, it appears both pathways develop simultaneously in the cotyledons but the CAM pathway, due to anatomical constraints, may be slower to develop than the C4 pathway. Cotyledons showed the ancestral Atriplicoid leaf anatomy, which leads to the question: Could a CAM cell be the precursor to the C4 pathway? Further study of this may lead to understanding into the evolution of C4 photosynthesis in the Portulaca.