Pseudomonas putida as saviour for troubled Synechococcus elongatus in a synthetic co-culture - interaction studies based on a multi-OMICs approach.

In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic co-culture of the cyanobacterium S. elongatus cscB, which supplies sucrose to the heterotrophic P. putida cscRABY, is investigated to identify potential interactions. Initial experiments reveal a remarkable growth-promoting effect of the heterotrophic partner on the cyanobacterium, resulting in an up to 80% increase in the growth rate and enhanced photosynthetic capacity. Vice versa, the presence of the cyanobacterium has a neutral effect on P. putida cscRABY, highlighting the resilience of pseudomonads against stress and their potential as co-culture partners. Next, a suitable reference process reinforcing the growth-promoting effect is established in a parallel photobioreactor system, which sets the basis for the analysis of the co-culture at the transcriptome, proteome, and metabolome levels. In addition to several moderate changes, including alterations in the metabolism and stress response in both microbes, this comprehensive multi-OMICs approach strongly hints towards the exchange of further molecules beyond the unidirectional feeding with sucrose. Taken together, these findings provide valuable insights into the complex dynamics between both co-culture partners, indicating multi-level interactions, which can be employed for further streamlining of the co-cultivation system.

LFQRatio: A Normalization Method to Decipher Quantitative Proteome Changes in Microbial Coculture Systems.

The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity, and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing. However, current workflows and methodologies are not suitable for simple artificial coculture systems where strain ratios are dynamic. Here, we established a workflow for coculture proteomics using an exemplar system containing two members, Azotobacter vinelandii and Synechococcus elongatus. Factors affecting the quantitative accuracy of coculture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated at the protein and cell level. We propose a new normalization method, named "LFQRatio", to reflect the relative contributions of two distinct cell types emerging from cell ratio changes during cocultivation. LFQRatio can be applied to real coculture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.

Class I Polyhydroxyalkanoate (PHA) Synthase Increased Polylactic Acid Production in Engineered Escherichia Coli.

Polylactic acid (PLA), a homopolymer of lactic acid (LA), is a bio-derived, biocompatible, and biodegradable polyester. The evolved class II PHA synthase (PhaC1 Ps6-19) was commonly utilized in the de novo biosynthesis of PLA from biomass. This study tested alternative class I PHA synthase (PhaC Cs ) from Chromobacterium sp. USM2 in engineered Escherichia coli for the de novo biosynthesis of PLA from glucose. The results indicated that PhaC Cs had better performance in PLA production than that of class II synthase PhaC1 Ps6-19. In addition, the sulA gene was engineered in PLA-producing strains for morphological engineering. The morphologically engineered strains present increased PLA production. This study also tested fused propionyl-CoA transferase and lactate dehydrogenase A (fused Pct Cp /LdhA) in engineered E. coli and found that fused Pct Cp /LdhA did not apparently improve the PLA production. After systematic engineering, the highest PLA production was achieved by E. coli MS6 (with PhaC Cs and sulA), which could produce up to 955.0 mg/L of PLA in fed-batch fermentation with the cell dry weights of 2.23%, and the average molecular weight of produced PLA could reach 21,000 Da.

The Preparation and Biomedical Application of Biopolyesters.

Biopolyesters represent a large family that can be obtained by polymerization of variable bio-derived hydroxyalkanoic acids. The monomer composition, molecular weight of the biopolyesters can affect the properties and applications of the polyesters. The majority of biopolyesters can either be biosynthesized from natural biofeedstocks or semi-synthesized (biopreparation of monomers followed by the chemical polymerization of the monomers). With the fast development of synthetic biology and biosynthesis techniques, the biosynthesis of unnatural biopolyesters (like lactate containing and aromatic biopolyesters) with improved performance and function has been a tendency. The presence of novel preparation methods, novel monomer composition has also significantly affected the properties, functions and applications of the biopolyesters. Due to the properties of biodegradability and biocompatibility, biopolyesters have great potential in biomedical applications (as implanting or covering biomaterials, drug carriers). Moreover, biopolyesters can be fused with other functional ingredients to achieve novel applications or improved functions. This study summarizes and compares the updated preparation methods of representative biopolyesters, also introduces the current status and future trends of their applications in biomedical fields.

Biosynthesis and biotechnological application of non-canonical amino acids: Complex and unclear.

Compared with the better-studied canonical amino acids, the distribution, metabolism and functions of natural non-canonical amino acids remain relatively obscure. Natural non-canonical amino acids have been mainly discovered in plants as secondary metabolites that perform diversified physiological functions. Due to their specific characteristics, a broader range of natural and artificial non-canonical amino acids have recently been applied in the development of functional materials and pharmaceutical products. With the rapid development of advanced methods in biotechnology, non-canonical amino acids can be incorporated into peptides, proteins and enzymes to improve the function and performance relative to their natural counterparts. Therefore, biotechnological application of non-canonical amino acids in artificial bio-macromolecules follows the central goal of synthetic biology to: create novel life forms and functions. However, many of the non-canonical amino acids are synthesized via chemo- or semi-synthetic methods, and few non-canonical amino acids can be synthesized using natural in vivo pathways. Therefore, further research is needed to clarify the metabolic pathways and key enzymes of the non-canonical amino acids. This will lead to the discovery of more candidate non-canonical amino acids, especially for those that are derived from microorganisms and are naturally bio-compatible with chassis strains for in vivo biosynthesis. In this review, we summarize representative natural and artificial non-canonical amino acids, their known information regarding associated metabolic pathways, their characteristics and their practical applications. Moreover, this review summarizes current barriers in developing in vivo pathways for the synthesis of non-canonical amino acids, as well as other considerations, future trends and potential applications of non-canonical amino acids in advanced biotechnology.

Systematic Engineering for Improved Carbon Economy in the Biosynthesis of Polyhydroxyalkanoates and Isoprenoids.

With the rapid development of synthetic biology and metabolic engineering, a broad range of biochemicals can be biosynthesized, which include polyhydroxyalkanoates and isoprenoids. However, some of the bio-approaches in chemical synthesis have just started to be applied outside of laboratory settings, and many require considerable efforts to achieve economies of scale. One of the often-seen barriers is the low yield and productivity, which leads to higher unit cost and unit capital investment for the bioconversion process. In general, higher carbon economy (less carbon wastes during conversion process from biomass to objective bio-based chemicals) will result in higher bioconversion yield, which results in less waste being generated during the process. To achieve this goal, diversified strategies have been applied; matured strategies include pathway engineering to block competitive pathways, enzyme engineering to enhance the activities of enzymes, and process optimization to improve biomass/carbon yield. In this review, we analyze the impact of carbon sources from different types of biomass on the yield of bio-based chemicals (especially for polyhydroxyalkanoates and isoprenoids). Moreover, we summarize the traditional strategies for improving carbon economy during the bioconversion process and introduce the updated techniques in building up non-natural carbon pathways, which demonstrate higher carbon economies than their natural counterparts.

Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production.

With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalkanoate (PHA) can be naturally produced by many species of bacteria and the PHA synthase is believed to be key enzyme in this natural pathway. Natural PHA synthases are diverse and can affect the properties of the produced PHAs, such as monomer composition, molecular weights, and material properties. Moreover, recent studies have led to major advances in the searching of PHA synthases that display specific properties, as well as engineering efforts that offer more efficient PHA synthases, increased PHA compound production, or even novel biopolyesters which cannot be naturally produced. In this article, we review the updated information of natural PHA synthases and their engineering strategies for improved performance in polyester production. We also speculate future trends on the development of robust PHA synthases and their application in biopolyester production.

Enzymatic process optimization for the in vitro production of isoprene from mevalonate.

BACKGROUND: As an important bulk chemical for synthetic rubber, isoprene can be biosynthesized by robust microbes. But rational engineering and optimization are often demanded to make the in vivo process feasible due to the complexities of cellular metabolism. Alternative synthetic biochemistry strategies are in fast development to produce isoprene or isoprenoids in vitro. RESULTS: This study set up an in vitro enzyme synthetic chemistry process using 5 enzymes in the lower mevalonate pathway to produce isoprene from mevalonate. We found the level and ratio of individual enzymes would significantly affect the efficiency of the whole system. The optimized process using 10 balanced enzyme unites (5.0 µM of MVK, PMK, MVD; 10.0 µM of IDI, 80.0 µM of ISPS) could produce 6323.5 µmol/L/h (430 mg/L/h) isoprene in a 2 ml in vitro system. In a scale up process (50 ml) only using 1 balanced enzyme unit (0.5 µM of MVK, PMK, MVD; 1.0 µM of IDI, 8.0 µM of ISPS), the system could produce 302 mg/L isoprene in 40 h, which showed higher production rate and longer reaction phase with comparison of the in vivo control. CONCLUSIONS: By optimizing the enzyme levels of lower MVA pathway, synthetic biochemistry methods could be set up for the enzymatic production of isoprene or isoprenoids from mevalonate.

The metabolism and biotechnological application of betaine in microorganism.

Glycine betaine (betaine) is widely distributed in nature and can be found in many microorganisms, including bacteria, archaea, and fungi. Due to its particular functions, many microorganisms utilize betaine as a functional chemical and have evolved different metabolic pathways for the biosynthesis and catabolism of betaine. As in animals and plants, the principle role of betaine is to protect microbial cells against drought, osmotic stress, and temperature stress. In addition, the role of betaine in methyl group metabolism has been observed in a variety of microorganisms. Recent studies have shown that betaine supplementation can improve the performance of microbial strains used for the fermentation of lactate, ethanol, lysine, pyruvate, and vitamin B12, during which betaine can act as stress protectant or methyl donor for the biosynthesis of structurally complex compounds. In this review, we summarize the transport, synthesis, catabolism, and functions of betaine in microorganisms and discuss potential engineering strategies that employ betaine as a methyl donor for the biosynthesis of complex secondary metabolites such as a variety of vitamins, coenzymes, and antibiotics. In conclusion, the biocompatibility, C/N ratio, abundance, and comprehensive metabolic information of betaine collectively indicate that this molecule has great potential for broad applications in microbial biotechnology.